This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.Calcium (Ca Their molecular weight  and, for some probes, the binding to nuclear structures prevent their diffusion into the cytosol through NPCs. Different GECIs have been developed in recent years . Indeed, whenever the signal-to-noise ratio (SNR) of the two probes at high [Ca2+] was directly assessed  in intact cells  or in the absence of external Ca2+ . In accordance with our previous observations . To evaluate the Kd, HeLa cells expressing H2BD3mCerulean3+16 were permeabilized with digitonin (100 µM) in an intracellular medium in the absence of external Ca2+ and energy sources and containing 600 µM EGTA. The cells were then exposed to different Ca2+ concentrations ranging from 20 nM to 3 mM  [ds were then exploited to estimate the [Ca2+] of the peaks reached in the nucleus upon maximal release of Ca2+ from the ER induced by IP3R (IP3 receptor) stimulation and SERCA (sarco/endoplasmic reticulum ATPase) blockade (2+ entry (SOCE) activation  were practically indistinguishable, while the following oscillations were asynchronous , penicillin (100 U/mL), and streptomycin (100 µg/mL), in a humidified atmosphere containing 5% CO2. Cells were seeded onto 18 mm diameter glass coverslips, and transfection was performed at 60–70% confluence using TransIT®-LT1 transfection reagent  with 1 µg of cDNA. Fluorescence and confocal imaging were usually performed 24 h after transfection.HeLa cells were grown in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal bovine serum, supplemented with For TO-PRO-3 staining, after fixation with 4% paraformaldehyde (PFA) for 10 min, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 min and washed and incubated with the dye  for 15 min. After PBS washing, coverslips were mounted using Mowiol . Images were collected at a Leica TCS SP5 II confocal system equipped with a PlanApo 100×, numerical aperture (NA) 1.4 objective. For all images, pinhole was set to 1 Airy unit. Cameleon donor excitation was performed with an argon laser line (458 nm), and a white laser at 512 or 643 nm was used to excite the Cameleon acceptor or TO-PRO-3 dye, respectively. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image with a 200 Hz acquisition rate. ImageJ program  was used for image analysis. To compare the absolute fluorescence of ECFP and mCerulean3, the mean fluorescence intensity measured within a ROI was normalized to the cpV mean fluorescence recorded in the same ROI, maintaining the recording parameters for each channel constant in subsequent acquisitions. The presence of cpV in both probes allowed the exploitation of its fluorescence as a parameter to normalize for the protein expression level in each cell analyzed.2, 0.4 KH2PO4, 1 MgSO4, 20 HEPES, 11 glucose, with pH 7.4 at 37 °C. The intracellular medium (IC) contained (in mM) 130 KCl, 10 NaCl, 1 MgCl2, and 20 HEPES, with pH 7.0 at 37 °C. All media were perfused through a temperature controller  to maintain a temperature of 37 °C in the chamber. Preincubations were also performed at 37 °C.Live cells expressing the fluorescent probes were analyzed using a DM6000 inverted microscope  with a 40× oil objective . LED excitation light  was properly filtered at 410 nm through a band-pass filter, and the emitted light was collected through a beam splitter  and HQ 535/30M (for cpV); dichroic mirror 515 DCXR). All filters and dichroic optics were from Chroma Technologies . Images were acquired using an IM 1.4C cool camera  attached to a 12-bit frame grabber. Synchronization of the excitation source and the camera was ensured through a control unit run by a custom-made software package, Roboscope . Exposure time varied from 50 to 200 ms depending on the intensity of the fluorescent signal of the cells analyzed. Images were usually acquired every second, with the only exception being calibration experiments, in which one image was acquired every 5 s. During the experiment, the coverslip was placed into an open-topped chamber and cells were maintained in the proper medium. The extracellular medium (EC) was modified Krebs–Ringer buffer (mKRB), which contained (in mM) 135 NaCl, 5 KCl, 1 MgCl(1)2+ release protocol: Classical experiments started in EC containing 1 mM CaCl2; after perfusion with 600 µM EGTA, cells were stimulated by perfusing with 100 µM histamine and 20 µM CPA, when indicated.ER-Ca(2)2; after 7 min of perfusion with EC containing 600 µM EGTA in the presence of 100 µM histamine and 20 µM CPA to empty the stores and maximize STIM1 oligomerization and translocation, cells were perfused with EC containing 2 mM CaCl2.SOCE-activating protocol: Classical experiments started in EC containing 1 mM CaClFunctionality experiments:min and Rmax values were obtained upon permeabilization of the cells in Ca2+-free IC medium containing 600 µM EGTA and 100 µM digitonin for 60 s. The Rmin was achieved by perfusing permeabilized cells with IC medium containing 600 µM EGTA, while the Rmax was achieved with IC medium containing 3 mM CaCl2. Alternatively, in intact cells, Rmin was obtained by incubation for 30 min at 37 °C in EC in the presence of 600 µM EGTA and 5 µM BAPTA-AM, while Rmax was obtained by adding 5 µM ionomycin in EC containing 3 mM CaCl2. The dynamic range was calculated by dividing Rmax (cpV/ECFP or cpV/mCerulean3 fluorescence intensity at saturated [Ca2+]) by Rmin .Dynamic range: In HeLa cells expressing the nuclear probes, R2+-free medium containing 600 µM EGTA, and basal FRET, which was obtained by incubating intact cells in EC in the presence of physiological 1 mM CaCl2.Photostability experiments: HeLa cells expressing each of the nuclear probes were illuminated every second using the same LED intensity at 410 nm. Images were acquired for 20 min at 1 Hz in two different FRET conditions: minimal FRET, which was obtained by bathing cells prepermeabilized with 100 µM digitonin in IC Ca2+-free medium containing 600 µM EGTA for 60 s and then incubated in the same medium without digitonin. Cells were then perfused with an IC at pH 7 containing known Ca2+ concentrations. At the end of each experiment, a saturating CaCl2 concentration (3 mM) was applied. IC supplied with 500 µM BAPTA free acid  was used to achieve [Ca2+] < 0.5 μM for Kd evaluation. The free [Ca2+] was estimated using MaxChelator2.5 and checked by fluorimetric measurements with Fura-2. The data obtained were plotted as log10[Ca2+] (x-axis) and R% (y-axis) and fitted using Origin 8 SR5 (OriginLab Corporation). Fitting was performed using the following equation:In situ calibration: HeLa cells expressing H2BD3mCerulean3+16 were permeabilized with 100 µM digitonin in IC Ca2+] of the IC medium used for the calibration experiments, 0.5 µM of Fura-2  was added to the solution and fluorescence was evaluated using the Perkin Elmer LS50B spectrofluorometer equipped with magnetic stirring  at 37 °C. The [Ca2+] concentration was calculated as follows: [Ca2+]free = 224 × Q [(R − Rmin)/(Rmax − R)], where 224 nm is the Fura-2 Kd, R represents the fluorescence intensity ratio Fλ1/Fλ2, in which λ1 is ~340 nm and λ2 is ~380 nm. R corresponding to the titration end points is denoted by the subscripts indicating the minimum and maximum [Ca2+]. Q is the ratio of Fmin to Fmax at λ2 (~380 nm).To evaluate the [CaRestriction and modification enzymes were purchased from Thermo Fisher Scientific . Digitonin, histamine, CPA, DMEM, trypsin, L-glutamine, and penicillin were purchased from Sigma-Aldrich . BAPTA and Fura-2 were purchased from Invitrogen, Thermo Fisher Scientific . All other materials were analytical or of the highest available grade.530/F430).Off-line analysis of FRET experiments was performed with ImageJ software . cpV and ECFP/mCerulean3 images were subtracted of background signals and distinctly analyzed after selecting proper ROIs on each cell; subsequently, a ratio between cpV and ECFP (or mCerulean3) emissions was calculated  and R is the R value at each time (t) of the experiment;A normalized R/R-min)/(Rmax − Rmin) × 100;R% is calculated as R% = . Statistical significance (p < 0.05) was determined by Student’s t-test (two-sided) for comparison of two groups with normal distribution and Wilcoxon test for comparison between two groups with non-normal distribution.All data are representative of at least three different experiments. Unless otherwise stated, numerical values presented throughout the text refer to mean ± SEM (


Noble TMDCs, unlike standard TMDCs such as MoS2 and WS2, operate in the ultrawide spectral range from ultraviolet to mid-infrared wavelengths; however, their properties remain largely unexplored. Here, we measured the broadband (245–3300 nm) optical constants of ultrathin PtS2 and PtSe2 films to eliminate this gap and provide a foundation for optoelectronic device simulation. We discovered their broadband absorption and high refractive index both theoretically and experimentally. Based on first-principle calculations, we also predicted their giant out-of-plane optical anisotropy for monocrystals. As a practical illustration of the obtained optical properties, we demonstrated surface plasmon resonance biosensors with PtS2 or PtSe2 functional layers, which dramatically improves sensor sensitivity by 60 and 30%, respectively.Noble transition metal dichalcogenides (TMDCs) such as PtS Howevere, or Te , making e, or Te , which s2, where M = Pt or Pd and X = S, Se, or Te) stands out, owing to their widely tunable bandgap from visible (for monolayers) to mid-infrared (for few-layers) spectral intervals [n and extinction coefficient k), which are crucial for predicting the performance of optoelectronic devices. Furthermore, these works [As a result, approximately 5000 potentially useful two-dimensional materials have recently been found . Among tntervals , high elntervals  and remantervals ,20. Broantervals , lasing ntervals , field-entervals , label-fntervals , holograntervals , and ultntervals , for exantervals ,28, absontervals ,29, and ntervals ,31 of grntervals ,32,33,34se works ,32,33,342 and PtSe2, which are typical representatives of group-X TMDCs. Through spectroscopic ellipsometry measurements, we accurately retrieved their broadband optical constants from ultraviolet to mid-infrared wavelengths (from 245 to 3300 nm). For all of the measured wavelengths, we discovered that PtS2 and PtSe2 exhibit non-zero extinction coefficients (k > 0), which explains recent advances of PtS2 and PtSe2 in photodetection [n ~ 4) of these materials, which makes them perfect candidates for all-dielectric nano-photonics [This work focuses on the optical properties of atomically thin PtSetection  for theshotonics ,36,37.2 and PtSe2 multilayers were purchased from 2d Semiconductors, Inc. . The samples were grown on c-cut sapphire substrates by chemical vapor deposition (CVD) using the highest purity (6N) gases (N2/H2) and precursors (S powder and Pt films) in semiconductor-grade facilities with subsequent water-assisted transfer on a 300 nm SiO2/Si substrate.Full area coverage PtSThe experimental setup used for Raman measurements was a Horiba LabRAM HR Evolution confocal scanning Raman microscope . All measurements were carried out using linearly polarized excitation at wavelength 632.8 nm; 1800 lines/mm diffraction grating, and × 100 objective (N.A. = 0.90), whereas we used unpolarized detection to have a significant signal-to-noise ratio. The spot size was approximately 0.43 µm. The Raman spectra were recorded with 0.75 mW and an integration time of 10 s at each point. The statistics were collected with 15 points for each sample, and the observed variation of the intensity for the spectra was less than 5%.−9 mBar) with a monochromatic Al-Kα X-ray source (1486.6 eV). Photoelectron spectra were acquired using fixed analyzer transmission (FAT) mode with 50 eV pass energy. The spectrometer energy scale was calibrated using C1s line position at 284.5 eV.The chemical state of the elements in the film was analyzed by X-ray photoelectron spectroscopy (XPS) in the Theta Probe tool  under ultrahigh vacuum conditions  using AFM in peak-force mode under ambient conditions. AFM measurements were carried out using ETALON, HA_NC silicon tips from TipsNano  with a spring constant of 3.5 N/m, a head curvature radius < 10 nm and a resonant frequency of 140 kHz. Images of PtS2 and PtSe2 surfaces were taken over a 3 × 3 µm2 area with a scan rate of 0.2 Hz; after that, data were analyzed by Gwyddion software.The thickness and surface morphology of PtS2 and PtSe2 were captured by an optical microscope  with a digital camera DS-Fi3.The surface images (2400 × 2400 pixels) of PtS2 and PtSe2 films surfaces in detail within different areas using a 1960 × 1280 pixel scan.A scanning electron microscope JEOL JSM-7001F  accompanied by a Schottky emitter in secondary electron imaging mode with a voltage of 30 keV and current of 67 µA, and a working distance of approximately 6.3 mm, was used to study surface features and homogeneity of PtSα radiation λ = 0154 nm was used to characterize the crystalline structure and phase of PtS2 and PtSe2 films. The XRD pattern was taken at ambient conditions by 2θ-scan over the range of 20–75° with a step of 0.05° and accumulation time of 2 s.An X-ray powder diffractometer  equipped with Cu KRstr/Rref, where Rstr is the reflection measured from the structures with films and Rref is the reference from a silver mirror NT64−114  that exhibits an average reflection of 99% between 350 and 1100 nm of light wavelength.The spectroscopic reflection analysis was performed in the 400–975 nm spectral range on a Biolam M-1 microscope  equipped with a 24 V, 100 W halogen light source and a QE65000 fiber-coupled grating spectrometer (Ocean Optics). The reflected light was collected in a backscattering configuration using an objective with magnification 100× (NA = 0.80). The experimental data represent the reflection ratio We used a variable-angle spectroscopic ellipsometer  with a single chamber monochromator with two gratings: 1200 g/mm for visible light (245–1040 nm) with 4.6 nm bandwidth and 600 g/mm for the infrared interval (1040–3300 nm) with 9.2 nm bandwidth. Spectroscopic ellipsometry was conducted over a wide wavelength range (from 245 to 3300 nm in steps of 1 nm) and multiple angles of incidence in the range of 30° to 80° with a step size of 5°.2 and PtSe2, we measured 11 elements of the Mueller matrix  on an Accurion nanofilm_ep4 ellipsometer  at 532 nm and 50° incident angle in rotation compensator mode.To investigate the in-plane anisotropic response of PtS2 and PtSe2 were calculated using density functional theory (DFT) within the generalized gradient approximation [2 were a = b = 0.3537 nm, c = 0.5019 nm, α = β = 90°, γ = 120°, and a = b = 0.3731 nm, c = 0.5072 nm, α = β = 90°, γ = 120° for PtSe2 [2 and PtSe2 were relaxed until the interatomic forces were less than 10−3 eV/Å, and a one-electron basis set was obtained from standard DFT calculations. Second, the real and imaginary parts of the frequency-dependent dielectric function were calculated using the GW approximation [The optical properties of PtSximation   on c-cut sapphire with subsequent water-assisted transfer on a 300 nm SiO2/Si substrate [2 and PtSe2 grow in the thermodynamically favored 1T-phase, as illustrated in 2 and PtSe2 have a thickness of 5 nm determined by atomic force microscopy (AFM). Therefore, our films have ten layers, since the interlayer distance in PtS2 and PtSe2 is 0.5 nm [2 and PtSe2 Raman modes Eg and A1g, corresponding to in-plane and out-of-plane vibrations of chalcogen atoms , respectively [g ~ 300 cm−1 and A1g ~ 335 cm−1 for PtS2; Eg ~ 175 cm−1 and A1g ~ 205 cm−1 for PtS2) corresponds to few-layer PtS2 and PtSe2 [2 and PtSe2 at the 632.8 nm excitation wavelength. Moreover, our samples uniformly cover the substrate as confirmed by optical and scanning electron microscopy (SEM) images for PtS2 and PtSe2 in 2/Si substrate. Nevertheless, these leftovers cover less than 5% of the surface and, hence, have a negligible effect on the resulting optical constants of PtS2 and PtSe2 studied here [2 and PtSe2 [2 and PtSe2 films was shown by measuring the X-ray diffraction (XRD) spectra displayed in the inset of Atomically thin PtSubstrate  to facils 0.5 nm . Raman sectively . Indeed,nd PtSe2 ,28 in agnd PtSe2 ,28 on Raied here . Additiond PtSe2 ,28. Fina2 and PtSe2, we performed spectroscopic ellipsometry (SE) measurements at multiple incident angles (30–80° in 5° steps) and wavelengths (245–3300 nm in 1 nm steps). The experimental scheme of SE setup is displayed in 2 and PtSe2. First, we checked the in-plane anisotropy of our samples using the Mueller matrix method [2 and PtSe2 in agreement with the previous study [2 and PtSe2.To properly quantify broadband optical properties of atomically thin PtS Ψ and Δ b–e, whicx method , in whicus study . In addi samples , which r2 and PtSe2 optical constants  in the Kretschmann [2 or PtSe2 as functional layers. In this scheme, the change in refractive index of a biological sample is detected, which involves monitoring the resonant reflection shift of the minimum (2 and PtSe2 films considerably enhance the structure sensitivity by 60 and 30% (2 or PtSe2) of about 4 nm with maximum refractive index sensitivity, as seen in 2 and PtSe2 improves device performance and, hence, could be used in numerous applications in optoelectronics and photonics.To highlight PtStschmann  configur minimum a. As see and 30% a,b, resp2 and PtSe2 films. We unveiled their ultrawide absorption and strong dielectric response, explaining the recent technological advancement of PtS2 and PtSe2-based optoelectronic devices. Moreover, we confirmed our PtS2 and PtSe2 optical constants both theoretically  and experimentally (reflectance measurements). Finally, we demonstrated that PtS2 and PtSe2 could serve as a functional layer in biosensors based on surface plasmon resonance. Altogether, these findings provide a foundation for PtS2 and PtSe2 optoelectronic and photonic devices, including label-free sensors [In conclusion, we report broadband 245–3300 nm) optical properties for atomically thin PtS5–3300 nm sensors , and ult sensors .


The short-circuit current density of 8.06 μA/cm2 and voltage of 34.63 V are 8.4 times and 3.5 times over that of pure Ti3C2Tx films, and 3.3 times and 4.3 times over that of commercial poly(tetrafluoroethylene) (PTFE) films, respectively. Furthermore, the fabricated TENG could be attached to human body to harvest energy from human motions, such as typing, texting, and hand clapping. The results demonstrate that the alternate-layered MXene composite nanosheet films through layer-by-layer stacking possess remarkably triboelectric performance, which broaden the choice of negative triboelectric materials and supply a new choice for high output TENG.The output power of the triboelectric nanogenerator (TENG) strongly depends on the performance of triboelectric materials, especially microstructures and functional groups of them. In this work, aiming at the excellent triboelectric ability, alternate-layered MXene composite films-based TENG with abundant fluorine groups(-F) through layer-by-layer stacking are designed and fabricated. Benefitting from the uniform intrinsic microstructure and increased dielectric constant, when the amount of the NbThe online version contains supplementary material available at 10.1186/s11671-021-03535-w. TENG based on the coupling effect of contact charging and electrostatic induction is considered as a powerful technology that effectively converts ambient mechanical energy into electrical energy –3. So fa2 , –CH3 [92 [3 .n+1XnTx, where M, X, and Tx represent the transition metals , C or N , and various surface end groups , respectively [3C2Tx nanofiber-based flexible TENG has been reported that the incorporation of Ti3C2Tx has significantly enhanced the dielectric property and thus improved the triboelectric output performance [3C2Tx served as a negative triboelectric material, which could be prepared by unidirectional freeze-drying and vacuum-assisted impregnation methods [3C2Tx/Ecoflex nanocomposite for universally energy harvest from various human motions [The MXene, as a new family of two-dimensional (2D) nanomaterials, is a novel type of layered transition metal carbides or nitrides which could be synthesized by selectively etching "A" elements from its precursor MAX phase . The genectively –14. The ectively . The incectively –18. All formance . Meanwhi methods . Cao et  motions .3C2Tx nanosheets containing a porous structure and interlayer spacers have been reported [However, like many other 2D materials, the performance of MXene is hindered due to its aggregation,  which rereported . Introdureported –27  into MX2CTx nanosheets are chosen as the spacer due to its higher electronegativity than carbon-based nanomaterials, and Ti3C2Tx serves as the bulk material owing to its high electronegativity. The prepared alternate-layered MXene composite nanosheet films can effectively reduce the self-restacking of Ti3C2Tx nanosheets and increase the interlayer spacing between Ti3C2Tx nanosheets, which will provide more effective nanochannels for -F group. It was found that such alternate-layered MXene composite nanosheet films-based TENG (AM-TENG) achieves the best performance with the weight ratio of 15% Nb2CTx. The maximum output current density and voltage are 8.06 μA/cm2 and 34.63 V, respectively, which are 8.4 times and 3.5 times over that of the pure Ti3C2Tx films and 4.1 times and 4.2 times over that of the commercial PTFE films. Additionally, the energy harvesting capability of alternate-layered MXene composite films-based TENG is demonstrated through capacitor charging. This work demonstrates a new type of triboelectric material for highly efficient green energy harvest.Here, layer stacked structure is adopted to design and fabricate alternate-layered MXene composite films with abundant -F group and uniform intrinsic microstructure. The Nb3AlC2 and Nb2AlC powders were purchased from Shandong Xiyan new materials technology Co., Ltd. Isopropylamine was provided by Shanghai Aladdin Bio-Chem Technology Co., LTD.All used chemicals were not further purificated. Ti3AlC2 was slowly added to (within 10 min) the above mixture under condition of continuous stirring. Afterwards, the reaction continued for one day under temperature of 35 ℃. Third, the prepared suspension was washed with deionized water for several times until its pH reached 6. Finally, the homogeneous Ti3C2Tx solution was sonicated under ice bath for 1 h and was further centrifuged for another 1 h at 3500 rpm. A total of 1 g of Nb2AlC powder was added gradually (within 5 min) into 10 mL of 50 wt% hydrofluoric solution. Then, the solution was constant stirred for two days at 35 °C to etch the Al layer from Nb2AlC. After centrifugation and repeatedly washing with deionized water, the collected sediments with the pH over 6 were dispersed in 10 mL isopropylamine solution for one day at room temperature for further intercalation. After centrifugation, the wet sediment was dispersed in 100 mL deionized water. Finally, after an 1 h centrifuge step at 3500 rpm rotating speed, the homogeneous Nb2CTx solution was obtained.Firstly, 1.6 g of LiF (Aladdin) was dissolved in 20 mL Hydrochloric acid  solution. Then, 1.0 g of TiThe TENG working under contact-separation mode was fabricated. First, a piece of copper foil was attached on an acrylic board to form a square shape electrode with a size of 1 cm × 1 cm (length × width). Then, 1 cm × 1 cm nylon film attached to the Cu foil was used as friction layer. Subsequently, the other counterpart with composite alternate-layered MXene composite film as friction layer was fabricated according to the same steps. Compared with the PTFE-TENG, the only difference is using alternate-layered MXene composite film instead of commercial PTFE films. The open-circuit output voltage, short-circuit current, and transfer charge of the alternate-layered MXene composite nanosheet films were measured by Keithley 6517B electrometers. Linear motor (Linmot E1100) was applied to provide an external periodic trigger at the frequency of 2 Hz.θ range from 5° to 60°) with Cu Kɑ radiation. The morphology of the nanosheets was confirmed by using scanning electron microscope , and energy-dispersive X-ray spectroscopy (EDS) mapping was performed on the same instrument (IXRF SYSTEMS). Raman (LABRAM HR EVOLUTION) spectra were acquired through a confocal Raman microscope with an excitation wavelength of 532 nm and a spectral grating of 1800 lines/mm. Spectra were acquired by focusing the laser through a 50 × objective. An LCR meter  was used to evaluate the dielectric constant of the nanosheets.The crystalline structure was characterized by a powder X-ray diffractometer  were on the surface of the outer Ti layer, which form a layered sandwich structure. Similarly, for Nb2CTx, niobium atoms filled the octahedral vertex position, assembling a layered ABAB structure. The observed Tyndall scattering effect in Fig. 3C2Tx solution and Nb2CTx solution had excellent stability and dispersity, which promised the uniform of each layer. Finally, the alternate-layered MXene composite nanosheet films were constructed through ABAB stacking under vacuum filtration  mapping pictures of the cross section are obtained. Nb, Ti, O, and F elements are detected throughout the scanned region, as shown in Fig. 3C2Tx and Nb2CTx nanosheets are stacked uniformly. In order to further analyze the material phases and the change of interlayer spacing between Ti3C2Tx and Nb2CTx nanosheets, X-ray diffractometer (XRD) measurements were conducted on pure Ti3C2Tx and alternate-layered MXene composite nanosheet films. As detailed in Additional file 3C2Tx film presents a strong (002) diffraction peak at 7.15°, which is consistent with previously reported results [2AlC MAX to 7.05° for Nb2CTx film due to the complete etching of Al atom layers [3C2Tx film and 5 wt% alternate-layered MXene composite nanosheet film shows that the intensity of the diffraction peak (002) obviously decreases, which indicates the introduction of Nb2CTx nanosheets. As the Nb2CTx contents increase from 10 to 15 wt%, changes in the diffraction angle gradually decrease, which means that the alternate-layered MXene composite nanosheet films interlayer spacing gradually increases due to the interaction between Nb2CTx nanosheets and Ti3C2Tx nanosheets. However, with the Nb2CTx content increasing from 20 to 25 wt%, the diffraction angle gradually increases from 0.6170 to 0.7536 nm . The XRD results are consistent with the SEM results. To further confirm the composition of alternate-layered MXene composite nanosheet films, Raman analysis was also performed. Figure 2CTx, Ti3C2Tx, and alternate-layered MXene composite nanosheet films with different Nb2CTx contents. The samples illustrate the expected vibrational modes for Ti3C2Tx , short-circuit current (Isc) density, and transferred charge density (Qsc). TENG based on the alternate-layered MXene composite nanosheet films with the same thickness was measured under the same conditions, as shown in Fig. Isc density, Voc, and Qsc of 15 wt% AM-TENG simultaneously increased remarkably compared with that of the pure Ti3C2Tx film. As the amount of the Nb2CTx increases to 15 wt%, the generated output Isc density, Voc, and Qsc of the AM-TENG gradually increase up to 8.06 μA/cm2, 34.63 V, and 11.19 nC, respectively, which are 8.4 times, 3.5 times, and 3.6 times over that of the pure Ti3C2Tx film , as described in Fig. 2CTx further increases from 15 to 25%, the Isc density, Voc, and Qsc decrease to 1.97 μA/cm2, 19.74 V, and 5.30 nC, respectively. Additional file Isc density, Voc, and Qsc with the gradient increase of Nb2CTx weight ratio.Figure 3C2Tx and alternate-layered MXene composite nanosheet films with different Nb2CTx concentrations and frequencies is shown in Fig. 3C2Tx film due to the formation of the microcapacitors interfacial network [3C2Tx and Nb2CTx likely aggregates, forming a conductive network and hence destroying the dielectric properties of alternate-layered MXene film. Therefore, leaking electricity may lead to a decrease in output performance [2CTx concentration, which has good consistence with the electrical results in Fig. 2CTx content further enhanced the triboelectric performance.For the contact-separation mode of the AM-TENG, the dielectric constant is an important parameter to determine the output performance. Therefore, the dielectric constant of alternate-layered MXene composite nanosheet films was characterized by complex permittivity model in the frequency range of 0.1 to 1000 MHz. Then, the dielectric constant of the Ti network . At highformance . The resIn order to further clarify the theoretical relationship between the output of AM-TENG and filler concentration, TENG can be reduced to a flat-panel capacitor model in Additional file Inside dielectric 1Inside dielectric 2x) of two triboelectric layers varies with mechanical force, and d1 and d2 are the thickness of dielectric material. Inside air gapThe voltage between the two electrodes can be given byV = 0Under short-circuit conditions and Equation  shows thIpp-sc of 8.65 μA/cm2, Voc of 37.63 V, and Qsc of 13.24 nC, respectively, is 4.3 times, 3.3 times, and 3.0 times over that of the commercial PTFE film. It illustrates that alternate-layered MXene composite nanosheet film is a promising triboelectric material. Figure 2CTx as a function of external load resistance ranging from 0.01 to 80 MΩ. Obviously, the short-circuit current density decreases with the increase of the connected external resistance, while the Voc follows an increasing trend. The instantaneous power of the TENG is obtained by calculating the measured load voltage and current density with the resistors. The corresponding peak power of the TENG is about 0.10 mW/cm2 under a load resistance of 5 MΩ (PTFE) film. In addition, the fabricated TENG can be attached to human body to harvest energy from simple human motions, such as typing, texting, and hand clapping. The results demonstrate that the alternate-layered MXene composite nanosheet films through layer-by-layer stacking can possess remarkably triboelectric performance, which enrich the triboelectric material family and supply a new choice for high output TENG.In summary, high-performance TENG based on alternate-layered MXene composite nanosheet films with abundant -F group through layer-by-layer stacking was successfully fabricated. The introduced NbAdditional file 1. Supporting Information.Additional file 2. SI video 1.Additional file 3. SI video 2.Additional file 4. SI video 3.Additional file 5. SI video 4.Additional file 6. SI video 5.Additional file 7. SI video 6.


Angiotensin-converting enzyme 2 (ACE2) is an important factor in coronavirus disease (COVID-19) interactions. Losartan (LOS) belongs to the angiotensin receptor blocker (ARB) family. Additionally, the protective role of ACE2 restored by LOS has been suggested and clinically examined in the treatment of COVID-19 patients. Furthermore, clinical trials with LOS have been conducted. However, the mechanism through which LOS enhances ACE2 expression remains unclear. In addition, the response of ACE2 to LOS differs among patients. Our LOS-treated patient data revealed a correlated mechanism of ACE2 with components of the renin-angiotensinogen system. We observed a significant positive regulation of MAS1 and ACE2 expression. In the context of LOS treatment of COVID-19, ACE2 expression could depend on LOS regulated MAS1. Thus, MAS1 expression could predict the COVID-19 treatment response of LOS. Losartan (LOS) belongs to the angiotensin receptor blocker (ARB) family3. The beneficial roles of ARBs or ACE blockers against COVID-19 were reported5. The LOS was suggested and clinically assessed in the treatment of COVID-19 patients 1. However, the response of ACE2 to LOS differed among studies. Carols et al. showed an increase in ACE2 levels with LOS6. It has been reported that LOS treatment does not affect changes in ACE27. The studies represent controversial response of LOS in context of ACE2.ACE2 is an important factor in coronavirus disease (COVID-19) interactions, because the virus uses the ACE2 receptor to gain entry into the cellIn this study, we identified the correlation analysis of RAS components after LOS treatment and analyzed the possible reasons for the individual differences in ACE2 expression in patients before and after LOS treatment. To analyze the correlation of ACE2 upregulation, public data were accessed using the NCBI Gene Expression Omnibus dataset (GEO database set) with the query dataset for GSE37824. The data were analyzed with GEO2R by defining groups for LOS baseline and LOS 6 week’s treatment in the LOS arm. The gene expression of samples was first assessed for the groups, normalized with GAPDH, and further analyzed for log2 (fold-change) in individual ACE2 expression to identify individual patient’s differences at baseline and 6 weeks of LOS-treated patients in the LOS arm.Figure 3. We analyzed renin angiotensin system (RAS) components AT1R, AGT, AT2R and MAS1 to evaluate the LOS mediated response. We observed 77% of patients showed decreased AT1R, and 23% showed increase AT1R log2 fold change from baseline Fig. MAS1) at 6 weeks of LOS treatment, compared to baseline , AT1R and AGT r = − 0.37), AT1R and AT2R (r = − 0.36) and AT1R and MAS1 (r = − 0.53) as shown in Fig. 7, AT1R aAGT and AT2R (r = 0.58) represented in Fig. ACE2 and AT2R (r = 0.46) shown in Fig. AGT and MAS1  as shown in Fig. A moderate correlation was observed for the gene pairs 2R r = 0.6 shown i2R r = 0.6 shown i2R r = 0.6 shown i9. This positive correlation of AGT and ACE2 with MAS1 could confirm the increased demand for ACE2 in LOS-treated patients.In this study, we identified the LOS regulated MAS1 expression. We observed significant positive correlations between AGT and MAS1 expression Fig. C and bet10. MAS1 expression was moderately negatively correlated with AT1R expression . Patients with peripheral artery disease were enrolled and randomized into 6 weeks of drug treatment vs baseline in LOS treated arm for paired analysis (n = 9). Patients underwent catheter excision of atherosclerotic tissue from one extremity at baseline and the contralateral extremity after treatment. 50 mg LOS once a day was given which reduced diastolic blood pressure by 4 mm Hg (p = 0.099). Sample were analyzed for expression profiling by array using Rosetta/Merck human 44 k 1.1 microarray platform. The gene expression of samples was first assessed for the groups, normalized with GAPDH, and further analyzed for log2 fold change in individual ACE2 expression through paired analysis at baseline and 6 weeks of treatment. The data were analyzed with GEO2R by defining groups for LOS baseline and LOS 6 week’s treatment in the LOS arm. The gene expression of samples was first assessed for the groups, normalized with GAPDH, and further analyzed for log2 fold change in individual ACE2 expression to identify individual patient’s differences at baseline and 6 weeks of LOS-treated patients in the LOS arm for gene ACE2, AT1R, AT2R, AGT and MAS1 for LOS treated samples.To analyze the mechanism of ACE2 upregulation, public data were accessed using the NCBI Gene Expression Omnibus dataset (GEO database set) with the query dataset for GSE37824 (Correlation analysis was performed using the R software. The correlation between LOS and AT1R, AGT, AT2R, or MAS1 was analyzed. The correlation between AT1R and ACE2, AGT, or AT2R was analyzed. In addition, the correlation between AGT and AT2R, between ACE2 and AT2R, between AGT and MAS1 was analyzed.Calculation of correlation coefficient were performed using R.Because we used NCBI Gene Expression Omnibus dataset to reveal the correlation between ACE2, ACE2, AT1R, AT2R, AGT and MAS1 for LOS treated samples, it is not required to identify the institutional and/or licensing committee approving the experiments, including any relevant details. In addition, it is also not required to declare documentation related to informed consent, ethics approval and donor organ/tissue source, including approved translations.


Accumulating evidence suggests that cholesterol accumulation in leukocytes is causally associated with the development of autoimmune diseases. However, the mechanism by which fatty acid composition influences autoimmune responses remains unclear. To determine whether the fatty acid composition of diet modulates leukocyte function and the development of systemic lupus erythematosus, we examined the effect of eicosapentaenoic acid (EPA) on the pathology of lupus in drug-induced and spontaneous mouse models. We found that dietary EPA supplementation ameliorated representative lupus manifestations, including autoantibody production and immunocomplex deposition in the kidneys. A combination of lipidomic and membrane dynamics analyses revealed that EPA remodels the lipid composition and fluidity of B cell membranes, thereby preventing B cell differentiation into autoantibody-producing plasma cells. These results highlight a previously unrecognized mechanism by which fatty acid composition affects B cell differentiation into autoantibody-producing plasma cells during autoimmunity, and imply that EPA supplementation may be beneficial for therapy of lupus. Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that affects multiple organs, and it is characterized by autoantibody production. As the concordance rate for SLE in identical twins is only 25-60%, this complex disease is caused by both genetic and environmental factors . PatientPolyunsaturated fatty acids are essential nutrients that affect chronic inflammatory diseases such as metabolic syndrome and cancer by regulating lipid metabolism and their immunomodulatory effects . Among o+ antigen-presenting cells (APCs) causes systemic autoimmunity in LXR-deficient mice by stimulating the production of the B cell growth factors, B cell activating factor (BAFF) and April, which support B cell expansion and autoantibody production -induced and spontaneous C57BL/6Information of key resources and primers for Q-PCR used in this study are shown in +/+ mice and C57BL/6Jlpr/lpr mice were purchased from Japan SLC, and female FVB/N mice were purchased from CLEA Japan. Eicosapentaenoic acid (EPA) ethyl ester was provided by Mochida Pharmaceutical. All animals were housed in a temperature-controlled animal room under 12 h light/12 h dark cycle under pathogen-free conditions. To induce lupus, FVB/N mice at 6 to 10 week of age were received a dayily topical dose of 25 mg of Beselna cream  or control vaseline on the right ear 3 times a week. The mice were fed fish meal-free diet supplemented with 5% palmitic acid or EPA for 6 weeks from 4 week-old. All of the animals were handled according to approved guide for the care and use of laboratory animals (ILAR Guideline). All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Nagoya University (No. 20253).Female C57BL/6JSpleens were digested with 1 mg/ml Collagenase type IV (Worthington) and 40 U/ml DNase I (Roche Diagnostics) in HBSS (−) at 37°C for 30 min. Single-cell suspension were incubated with anti-mouse CD16/32 antibody (BioLegend) to prevent nonspecific binding of antibodies and stained with fluorescence-conjugated antibodies in 0.5% BSA, 5 mM EDTA in PBS. The following conjugated antibodies were used for the staining: B220 (RA3-6B2), CD23 (B3B4), CD21 (7E9), CD138 (281-2), CD95 (SA367H8), GL7 (GL7), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11c (N418), MHCII (M5/144.15.2), PDCA (9.27E+02), CD11b (M1/70), Ly6G (1A8), CD44 (IM7), CD62L (MEL-14), PD-1 (29F.1A12), CD25 (3C7) (BioLegend), Peanut Agglutinin , and DAPI (BioLegend). For intracellular staining, cells were incubated with Transcription Factor Fix/Perm Working Buffer (TONBO Biosciences) at room temperature for 30 min. After washing twice with Flow Cytometry Perm Buffer (TONBO Biosciences), cells were stained with APC-conjugated anti-Blimp1  or PE-conjugated anti-Foxp3  at room temperature for 20 min in the dark. After washing, cells were analyzed on MACSQuant Analyzer (Miltenyi Biotec) and FlowJo software (BD Bioscience). An example of the gating strategy for B cells, T cells and myeloid cells is shown in Kidneys were collected and embedded in OCT compound and frozen on dry ice. Three μm frozen-sections were air-dried, fixed in acetone, blocked with 6% BSA and 4% normal goat serum in PBS, and stained with FITC-conjugated AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) and FITC-conjugated goat IgG fraction to mouse complement C3 . Sections were mounted using ProLong Gold Antifade Reagent with DAPI (Invitrogen). Evaluation of the fluorescence intensity of IgG and C3 was performed by scoring the intensity of staining for individual glomeruli as 0 (negative), 1 (positive above background), 2 (positive), and 3 (brightly positive) for at least 10 glomeruli per section. Spleens were collected and fixed with neutral buffered−formalin and embedded in paraffin. Five μm–thick sections were stained with hematoxylin and eosin. Images were obtained using BZ-X700 fluorescence microscope (KEYENCE).4 cells in each well of a 96-well plate and incubated at 37°C with 5% CO2 for 20 to 24 h. B16-Blue IFNα/β cell supernatant was incubated with QUANTI-Blue (InvivoGen) and secreted embryonic alkaline phosphatase (SEAP) levels were determined using a spectrophotometer at 620 nm.Serum levels of total IgG, total IgM (Invitrogen), autoantibodies against nuclear, histone  and dsDNA (FUJIFILM Wako Shibayagi), and BAFF (R&D Systems), were measured by ELISA kits. Cell-free culture medium was collected and the levels of IL12p70 (Invitrogen), IL6 and TNFα (R&D Systems) were measured by ELISA kits. IFNα/β amount in serum and culture medium was quantified using IFN sensor-B16-Blue IFNα/β cells  according to manufacturer’s instruction. Briefly, mouse serum diluted 1:10 or cell-free culture medium was combined with RPMI 1640 supplemented 10% FBS containing 2 × 106 cells were cultured in RPMI 1640 supplemented with 10% FBS, 15% GM-CSF conditioned medium, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM Sodium pyruvate, 50 μM 2-ME in 10 cm petri dish for 7 days. BMDCs (1 × 106 cells) were seeded per well in RPMI containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in 12-well plate and treated with ethanol, 50 μM palmitate  or 50 μM EPA  with 5 μM BSA overnight. Cells were then stimulated with 10 ng/ml LPS (Sigma) for 4 or 24 h or 5 ng/ml IFNγ (PeproTech) for 6 h. APCs, naïve B cells and pan-B cells were isolated using CD11c MicroBeads UltraPure, B Cell Isolation Kit and Pan B Cell Isolation Kit II (Miltenyi Biotec) respectively, according to the manufacturer’s instructions. For B cell differentiation assay, naïve B cells (5 × 104 cells) from spleen were seeded per well in RPMI 1640 containing 10% FBS, 100 U/mL penicillin, 100 mg/ml streptomycin and 50 μM 2-ME in 96-well plate and treated with 1 μg/ml anti-CD40 antibody  and 3 ng/ml recombinant murine IL4 (PeproTech) in the presence of ethanol, palmitate or EPA with 5 μM BSA for 96 h. Naïve B cells were also co-cultured with CD11c+ cells from spleen and stimulated with 1 μg/ml anti-CD40 antibody and 1 μg/ml R848 (AdipoGen) in the presence of ethanol or 50 μM EPA with 5 μM BSA for 96 h.Murine bone marrow-derived dendritic cells (BMDCs) were obtained as described previously . Bone ma36B4. Primer sequences are listed in Total RNA was isolated from cells and tissues with Sepazol  according to the manufacturer’s instruction. Five hundred ng of total RNA was used for cDNA synthesis, and gene expression was quantified by real-time PCR using SYBR Green and StepOne Plus Instrument (Applied Biosystems). Gene expression levels were normalized to m/z = 74, C18:1 (17.74 min) m/z = 69, C19:0 (18.59 min) m/z = 74, C20:4 (22.60 min) m/z = 79, C20:5 (23.74 min) m/z = 74, and C22:6 (26.97 min) m/z = 79.Cells were snap frozen in liquid nitrogen and subsequently subjected to Folch lipid extraction . Nonadec13C16-palmitic acid was dissolved in acetonitrile). The mixture was centrifuged at 20,000 × g for 10 min, and the organic phase was collected. The remaining precipitate and aqueous phase were then mixed with 400 μl of dichloromethane, and the resulting mixture was centrifuged at 20,000 × g for 5 min to give the organic phase. The combined organic phases were then evaporated to give the residues and dissolved in a solution consisting of 100 μl of a mixture of isopropyl alcohol/acetonitrile/water . UHPLC-Triple TOF/MS analyses were performed on a Shimadzu UHPLC Nexera X2 system (Shimadzu) using a TSKgel ODS-120H column  and a Triple TOF 5600+ (SCIEX) with an electrospray ionization device running in the positive and negative ion mode. The autosampler injection volume was set to 10 mL and the eluent flow rate to 0.4 ml/min. Mobile phase A consisted of a 0.1% (v/v) solution of formic acid and 10 mM ammonium formate in acetonitrile/water , and mobile phase B consisted of a 0.1% (v/v) solution of formic acid and 10 mM ammonium formate in isopropyl alcohol/acetonitrile . The linear gradient conditions were as follows: 40% B at 0 min, 43% B at 3 min, 55% B at 15 min, 99% B at 25 min, 99% B at 27 min and 40% B at 27.01 min, followed by a 2.99 min equilibration time. The detector conditions were as follows: ion spray voltage at 5500 V, source temperature of 350°C, ion source gas 1, 60 psi, ion source gas 2, 60 psi, declustering potential, 80 V and collision energies of 45 V, collision energy spread, 15 V. Nitrogen was used as the collision gas. The raw data “mzXML” was used to convert to “abf “ format with the ABF converter. The MS DIAL equipped with FiehnLib24 was used for raw peaks exaction, peak alignment, deconvolution analysis and identification.Cells were snap frozen in liquid nitrogen and subsequently subjected to lipid extraction with 200 μl of Ultrapure water (FUJI Film Wako), 200 μl of methanol (FUJI Film Wako), 400 μl of dichloromethane (FUJI Film Wako) and 20 μl internal standard solution mixture  and 10 μl of 100 μM 4 cells were plated onto 9.5 mm multi glass-bottom dishes in RPMI1640 containing 10% FBS 0.05% IL3 conditioned medium with 100 μM palmitate or 100 μM EPA and incubated for overnight. Cells were incubated with 10 μM Laurdan  at 37°C for 30 min. Spectral data were obtained with FV1000-D IX81 confocal laser scanning microscope (Olympus) at excitation 405 nm. The emission signal was collected in two bands: 430–455 nm and 490–540 nm. Spectral data were processed by the SimFCS software (Laboratory for Fluorescence Dynamics). GP value of each pixel was used to generate the pseudocolored GP image.Membrane dynamics was analyzed as described , 21. Bripost hoc Tukey-Kramer’s multiple comparison test for the comparison among 3 or more groups if homogeneity of variance and normality were confirmed, or Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test if not confirmed. Data are presented as the mean ± SEM. p < 0.05 was considered statistically significant.Data were analyzed using Prism Version 9 software (GraphPad). For data with two groups, unpaired t-tests were performed as homogeneity of variance and normality were confirmed. The data were analyzed by one-way analysis of variance (ANOVA) followed by the To elucidate the effect of EPA on the progression of SLE, we first induced lupus in wild-type FVB/N mice using imiquimod (IMQ), a TLR7 agonist, and prophylactically fed the mice a diet supplemented with 5% EPA or 5% palmitate as a control to match caloric intake Table S3To clarify what cell types are influenced by IMQ treatment and/or EPA supplementation, we next performed immunophenotyping of spleens using flow cytometry (lpr/lpr (B6lpr/lpr) mice, which carry a mutation in the apoptosis-inducing receptor gene Fas and develop lupus manifestations as early as 8 week-old  CD3+B220+ abnormal T cells in B6lpr/lpr mice was robust, and the T lymphoid cellularity was accordingly affected  and R848. Naïve B cells were differentiated into B220loCD138+ plasma cells in the presence of CD40L and R848, and the induction of plasma cells was further enhanced by coculture with larger numbers of CD11c+ cells  in the presence of vehicle, palmitate, or EPA , Xbp-1, and Irf4, which are essential transcription factors for plasma cell differentiation, whereas it did not affect the expression of Cd79b, a transmembrane protein that forms a complex with the B cell receptor (BCR) and that is expressed in all B lineage cells , a potent receptor for omega-3 fatty acids including EPA, and/or pro-resolving lipid mediators, e.g., resolvin E1 and E2, derived from EPA and their receptors  and contribute to the prevention of inflammation  and Japan Agency for Medical Research and Development (CREST) . This study was also supported by research grants from Smoking Research Foundation (TS), Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, Ono Medical Research Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, GSK Japan Research Grant 2018, SENSHIN Medical Research Foundation, Kao Research Council for the Study of Healthcare Science, Kowa Life Science Foundation, The Hori Sciences and Arts (AI), Aichi Kidney Foundation (AK). GSK was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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.


LaL1(TTA)3 reported herein exhibits 77 K phosphorescence observable by the naked eye for up to 30 s. Optical spectroscopy, density functional theory (DFT) and time-dependent DFT techniques have been applied to investigate the photophysical processes of this compound. In particular, on-off continuous irradiation cycles reveal a charging behaviour of the emission which is associated with triplet-triplet absorption because it shows a shorter rise lifetime than the corresponding decay lifetime and it varies with illumination intensity. The discovery of the behaviour of this compound provides insight into important photophysical processes of the triplet state of organo-lanthanide systems and may open new fields of application such as data encryption, anti-counterfeiting and temperature switching.Emission from the triplet state of an organo-lanthanide complex is observed only when the energy transfer to the lanthanide ion is absent. The triplet state lifetime under cryogenic conditions for organo-lanthanide compounds usually ranges up to tens of milliseconds. The compound  Compounds featuring long-lived luminescence have potential applications in a variety of fields, including anti-counterfeiting and switches. Here the authors report a lanthanide-based compound that exhibits phosphorescence observable by the naked eye for up to 30 s at 77 K; On-off continuous irradiation cycles reveal a charging behaviour associated with triplet-triplet absorption, showing a shorter rise lifetime than the decay lifetime. Light-harvesting techniques therefore employ coordination to organic molecules2, or doping into strongly-absorbing inorganic chromophore systems3. Organic complexes of lanthanide ions comprise a burgeoning research area with a wide spectrum of applications: optical and magnetic resonance imaging5, contactless thermometry6, drug delivery7, and organic light-emitting diodes8, for example. In optical applications, the properties of the triplet state , giving phosphorescence emission lifetimes in the range of up to hundreds of milliseconds16. However, this is an order of magnitude shorter than for the lanthanide compound reported herein with phosphorescence observable up to 30 s with the naked eye.Lanthanide ions 3 −1,3,5-triazin-2-yl)-N,N-diethylaniline)] tris)lanthanum (III)) is observable for up to 30 s at 77 K as illustrated 3 not only provides insights into its phosphorescence mechanism but also leads to a new dimension of understanding and application for lanthanide complexes. The ultra-long phosphorescence lifetime occurring in the cryogenic state means that this compound can be utilised to add an additional key step as an anti-counterfeiting agent, or as a low-temperature switch.In this work, we describe the long-lived phosphorescent organo-lanthanide compound. Under cryogenic conditions using laser excitation, the green luminescence of ted Fig. . BesidesLaL1(TTA)3 3 tris lanthanum(III), Supplementary Fig. 1H and 13C NMR spectra 3 and L1. The lanthanide ion complex LaL1(TTA)3 exhibits sharp, well-resolved spectra in both solvents, demonstrating stable coordination of the ligands with the La3+ ion. The ligand L1 peaks are observed in the FT-IR spectrum of LaL1(TTA)3 whereas the carbonyl stretching mode of TTA is shifted to lower energy in LaL1(TTA)3 showing the involvement in complex formation with La3+ 3 Fig.  and anotfluoro-1--thienyl-LaL1(TTA)3 do not exhibit significant change with concentration at room temperature 3 are remarkable. Upon irradiation at 77 K, 10 μM LaL1(TTA)3 in toluene shows a bright green afterglow which was observable up to 30 s by the naked eye under 355 nm laser excitation 3, exhibits a shorter phosphorescence decay and the emission is only observable up to 2 − 3 s by the naked eye while that of L1 is observed above 5 s 3 at 100 μM concentration 3 at 10 μM concentration in toluene 3 in toluene at 77 K when the concentration is raised above 10 μM. A reversible interconversion occurs (from form A) while cooling to another form (form B) which has the singlet emission transition with maximum intensity at 469 nm. The triplet T1 → S0 transition of form B is observed at 540 nm. The reversible interconversion occurs whilst cooling and both forms may be present at 77 K depending upon the concentration in toluene 3 at 100 μM concentration in toluene is achieved at a lower concentration by changing to the more polar solvent chloroform 3 in toluene at room temperature is red-shifted to give a broad singlet peak at 540 nm in the solid-state 3 complex. Turning to time-dependent density functional theory (TD-DFT), the first singlet state transition, S1 – S0, of the π−stacked conformation of L1 is calculated to be at lower energy compared to the isolated molecule L1 3 where the −TTA fragment is participating in the energy transition 3 from 250 K to 10 K, a new band at 560 nm is observed 3 at 77 K were determined from monoexponential fits to the decay profiles and were 0.18 s for L1 and between 3.5 s and 4.3 s for LaL1(TTA)3, depending upon the solvent 3 was measured as 0.18 s, which is shorter than the phosphorescence decay lifetime of Phen itself (1.6 s26). Hence the longer phosphorescence lifetime of LaL1(TTA)3 compared to that of LaPhen(TTA)3 does not solely arise from metal coordination but involves other photophysical processes.The phosphorescence lifetimes of ent Fig. . The lifent Fig. . For comLaL1(TTA)3 was performed using the kinetics module as illustrated in Fig. LaL1(TTA)3 at 10 μM concentration in toluene at 77 K revealed a distinct charging feature for this sample . The 505 nm and 540 nm emission bands correspond to triplet emission 3 mainly exists at 100 μM concentration in toluene. When monitoring the phosphorescence decay at different concentrations of LaL1(TTA)3 in toluene, the rise lifetime (τr) at 505 nm  did not change much with concentration, while the lifetime monitored at 540 nm  decreases with increasing concentration due to the change in relative amounts of A and B  slightly longer than the rise (charging) lifetimes (τr). The charging profile can be attributed to triplet-triplet absorption involving T1, where the decay lifetime is always longer than the charging lifetime as derived in Methods, Eq. (1. The detailed solution of the rate equations laid out in the Methods shows that the inclusion of triplet-triplet absorption leads to kr > kd for the rise (kr) and decay (kd) rate constants and that kr should exhibit a linear relation with charging intensity 3, where π-stacking interaction is much stronger, differ somewhat from those in toluene. However, when monitoring at 427 nm 3 in toluene  indicates a successive reduction in the population of the singlet state until all processes are in equilibrium. Since the volume available per formula unit is reduced by a factor of ~1 million on going from 10 μM LaL1(TTA)3 in toluene to solid-state LaL1(TTA)3, the heavy atom effect of La3+ in increasing the intersystem crossing rate via spin-orbit coupling is more pronounced in the solid-state. This is demonstrated in Supplementary Fig. 1 charging profile, where the only change from the parameters employed in Supplementary Fig. 1 charging profile is much faster in the solid-state, followed by a greater intensity decrease. The schematic photophysical mechanism for LaL1(TTA)3 at 10 μM concentration in toluene is illustrated in Fig. The observations when employing solid  nm Fig.  or 515 n nm Fig. , in both nm Fig. . Upon exene Fig. . The spiLaL1(TTA)3 in an appropriate vessel while the remaining squares are filled with a blue-emitting compound (L1 is used herein). The vessel shows blue emission from all squares at room temperature  were used as received without further purification.3·2H2O and L1 were prepared by the literature methods without modification28.La(TTA)N,N-diethylaniline  and cyanuric chloride  was heated at 75 °C for 24 h under nitrogen. After complete reaction, the mixture was extracted with 100 mL dichloromethane (30 mL × 3). The combined organic phase was dried over anhydrous MgSO4, then filtrated through celite and washed with dichloromethane (10 mL × 3). The resulting mixture was evaporated under vacuum and the solid residue was washed with 50 mL × 3 of n-hexane. Finally, the intermediate 4--N,N-diethylaniline was afforded from twice recrystallization in acetone as a yellow solid . Potassium  was added to a stirred solution of 3,5-dimethylpyrazole  in dry THF (60 mL) under N2. The resulting mixture refluxed until a colourless solution was formed. Subsequently, 4--N,N-diethylaniline 2  was added to aforementioned solution at about 10 °C. The reaction mixture was further stirred at room temperature for 0.5 h and then refluxed at 70 °C for 6 h. After complete reaction, the mixture was kept at 4 °C for 12 h (overnight) to ensure a complete precipitation. The precipitate was finally filtered, washed repeatedly with THF (10 mL × 3) and n-hexane (10 mL × 3). The solid was dried under vacuum to afford L1 as a yellow solid .A mixture of 3.2H2O was synthesized by the literature method29 with some modifications. Briefly, HTTA  was dissolved in 15 mL ethanol in a 150 mL flask, with stirring at room temperature. Then, the pH of the solution was adjusted to 6–7 by the addition of NaOH solution (1.0 M). After that, 1 mmol of LaCl3·7H2O  solution in 5.0 mL of deionized water was then added into the above mixture at 60 oC. Then deionized water (100 mL) was added into the above mixture with vigorous stirring for 2 h at 60 oC to ensure complete precipitation. After cooling to room temperature, the precipitate was filtered, washed repeatedly with water, and dried overnight under vacuum at room temperature to afford La(TTA)3.2H2O as a light yellow solid .La(TTA)L1  and La(TTA)3·2H2O  in THF (20 mL) was stirred at room temperature overnight. The solvent was then evaporated and the residue was re-dissolved in a minimum amount of diethyl ether and precipitated by n-hexane. The bright yellow solid was obtained by twice repeated precipitation.The solution of the 1H NMR  δ 8.24 , 7.48 , 7.37 , 7.00 – 6.93 , 6.70 , 6.13 , 6.11 , 3.43 , 2.88 , 2.49 , 1.22 ; 13C NMR  δ 180.17, 172.60, 169.88 , 162.41, 155.90, 152.23, 146.61, 145.05, 132.01, 131.03, 128.76, 127.58, 120.20, 119.12 , 113.31, 111.00, 91.56, 44.82, 16.91, 13.68, 12.62; 1H NMR  δ 8.10 , 7.17 , 6.80 , 6.49 , 6.45 , 6.40 , 5.63 , 2.81 , 2.79 , 2.54 , 0.80 ; 13C NMR  δ 180.60, 172.29, 170.13 , 162.15, 155.58, 151.87, 146.57, 144.76, 131.92, 130.80, 127.40, 120.63 , 120.49, 113.04, 110.68, 91.74, 44.13, 16.45, 13.75, 12.13. Elemental analysis : C, H, N , S.Yield: 71 %. 3 samples were synthesized by the literature method30. Typically, TTA (3 mmol) and Phen (1 mmol) were dissolved in 15 mL ethanol in a flask with stirring, at room temperature. Then, the pH of the solution was adjusted to 7.0 by the addition of NaOH solution (1.0 M). After that, 1 mmol of LaCl3 solution in 5.0 mL of deionized water was then added into the above mixture at 60 °C with vigorous stirring for 1.0 h to ensure complete precipitation. The precipitate was finally filtered, washed repeatedly with ethanol and water, and dried overnight under vacuum.The LaPhen(TTA)24H12F9LaNaO6S3+ [La(TTA)3 + Na]+ 824.8608, found 824.8608. HRMS (ESI) calculated for C24H14F9LaO8S3- [La(TTA)3 + COO]- 847.8698, found 847.867931. Elemental analysis : C , H , N .Yield: 65%; HRMS (ESI) calculated for C1H and 13C NMR spectra were collected on a Bruker Ultrashield 400 Plus 400 MHz NMR spectrometer. High-resolution mass spectra (HRMS) were obtained on an Agilent 6540 UHD AccurateMass Q-TOF LC/MS. Elemental analyses were performed by an Elementar microcube elemental analyzer. FT-IR ATR spectra were recorded by a Perkin Elmer® FT-IR Spectrum Two instrument.Both ® LAMBDA 1050+ UV/VIS/NIR double beam spectrophotometer. The emission, excitation spectra, and luminescence decay curves were recorded using a Horiba Fluorolog®-3 instrument with a 450 W xenon lamp. The signal was detected by a Hamamatsu R928 photomultiplier and corrected with excitation and emission correction factors to eliminate response characteristics. For laser irradiation, a Nd3+:YAG pulsed laser was used as the excitation source together with a third harmonic generator (10 mJ) and an optical parameter oscillator . A 10 mm pathlength cuvette was used for all room temperature measurements. A custom-made liquid nitrogen cryostat with an NMR tube sample holder was employed for 77 K studies. The Horiba kinetics module as shown in Fig. The ultraviolet-visible absorption spectra were measured in solution in the range 200–800 nm by a Perkin Elmer32. Molecules were built using Avogadro software33. The integration grid was set to Lebedev 590 points with the final grid of Lebedev 770 points. The nature of each stationary point was characterized using frequency calculations. Geometry optimizations and free-energy calculations were performed at the PBE0-D3/def2-TZVP level of theory34, with the Stuttgart in-core effective core potentials and basis sets for the lanthanide ions36. Solvation by toluene (ε = 8.93) was accounted for using the conductor-like polarizable continuum model (CPCM)37. TD-DFT calculations were performed with the same level of theory using the Pople solver. The simulated absorption spectra employed the bandwidth of 3000 cm−1. UCSF Chimera (version 1.14)38 was used for the visualization of geometries and orbitals.DFT calculations were performed using the ORCA 4.0 suite of programs0, S1, T1, and Tn. The rate constants (s−1) are as defined in Table We consider the four-level system comprising SThe rate equations are:1 and Tn (since decay of T1 is so slow) gives:A is a constant and the rise exponent kr > c = kd, where kd is the decay rate constant of T1 . The fit to the experimental data using this equation is shown in Supplementary Fig. A simple solution, considering the estimated values of rate constants, when applying the steady-state approximation to STM by the Rosenbrock stiff 3-4th order numerical method (or the Modified Extended Backward Differentiation Equation Implicit Method) with initial conditions that [S0(0)] = 1, [S1(0)] = 0, [T1(0)] = 0 and [Tn(0)] = 0 is straightforward and representative results are given in the Supplementary Fig. The numerical solution to Eqs. –4) usin usin4) uSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba- Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy . This reThe identification of EGFR as an oncogene has led to the development of anticancer therapeutics directed towards it . The useThe FDA (Food and Drug Administration) and EMA (European Medicines Agency) already approved several low molecular tyrosine kinase inhibitors  as first line treatment for sensitive EGFR-mutant patients with non-small-cell lung cancer (NSCLC) . AlthougO-methyl (2′-OMe) units], its human serum stability and toxic potential are increased in breast cancer, liver cancer, and malignant glioblastoma cells -labeled RNA fragment to trigger RNase H activity in comparison to the nonmodified reference ASO-22 . Only traces of shorter 6 nt long product was visible. These results indicate that RNA was cleaved within the internucleotide linkages marked by the arrows in the 5′-pUCG GGC U↓CU ↓GGA GGA AAA GAAA-3′ sequence, that is, between U and C or U and G units, respectively. The prolonged incubation time of the cleavage reaction up to 240 min , which is between a C and U. The obtained results also suggest that the rate of hydrolysis of 1/2 is slightly faster than that of ASO. This experiment shows the sufficient efficiency of the tested nanostructures for duplex formation with the target RNA, resulting in RNase H activation and successful degradation of the target RNA.Antisense oligonucleotides exert their gene silencing activity either by sequence-specific hybridization to target mRNA molecules and recruitment of RNase H, which cleaves the target RNA, as well as by other mechanisms involving the steric hindrance of mRNA ribosomal activity, or by altering mRNA maturation . Here, wnterpart . The fir 240 min  resultedproducts . Thus, w1/2 towards the endogenous mRNA of EGFR protein, we used human squamous cell carcinoma cells (A431) expressing higher levels of EGF receptor compared to HeLa  and MCF-7 (human breast cancer) cell lines -ate [Fe(C2B9H11)2]− (FESAN)) (50 and 100 nM). The 1,2-DCDDB boron cluster (100 nM) increased the content of cells in the S phase to 24% and strongly decreased the content of cells in the G2 phase to 2% compared to the control cells . The changes observed in a cell cycle phases ratio induced by ASO-22 and 1/2 follow a very similar pattern to 1,2-DCDDB, with small changes only in the S (increased by 3–14%) and G1 phases (up to 10%) compared to the control cells, while the abundance of the G2 phase remains very low in all experiments (2–3%) compared to 15% for the control cells. The most significant changes in the distribution of the cell cycle phases were observed for FESAN-treated A431 cells (100 nM). Here, the ratio of cells in the S phase increased to 74% and that in the G1 and G2 phases decreased to 24% and 2%, respectively.We also checked the influence of free boron clusters on the cell cycle by treating A431 cells with 1,2-dicarba-Colchicine and quercetin were used as positive controls for A431 cells. Earlier data reported that colchicine induces cell cycle arrest at the G2/M phase in MCF-7 cells , while q1/2 exert inflammatory responses, we stimulated LPS-primed human primary monocyte-derived macrophages with nanostructures 1/2, oligonucleotide ASO-22 and boron clusters 1,2-DCDDB and FESAN for 16 h and studied the release of major NLRP3 inflammasome-dependent pro-inflammatory cytokine IL-1β. As positive controls for IL-1β release, cells were stimulated with nigericin, an NLRP3 inflammasome-activating ionophore toxin derived from Streptomyces hygroscopicus -RNA. In the 5′-p*UCGGGC6U7C8U9GGAGGAAAAGAAA-3′ oligonucleotide, cleavage occurred after U9 and then after U7 in the presence of ASO-22, with only trace amounts of the product of cleavage after C6, while in the presence of 1/2, cleavage occurred after the same units  (see The silencing activity of nanostructures 8 h) see . The ant8 h) see . In rece1/2 are capable of activating the NLRP3 inflammasome. As shown in 1/2 containing 1,2-DCDDB did not stimulate primary macrophages isolated from two healthy human donors to produce IL-1β in contrast to FESAN, which induced IL-1β secretion at a level of approximately 30–50% of that of nigericin-induced IL-1β. These results show the lower pro-inflammatory properties of our compounds and demonstrate their improved potential safety in therapeutic application over boron carriers currently used in BNCT.Boron is a regulator of the immune and inflammatory reactions and macrophage polarization, playing an important role in augmenting host defense against infection, with possible roles in cancer and other diseases . Boron-cN,N-diisopropyl)]-phosphoramidite were purchased from Glen Research . FESAN was obtained from Katchem (Rež n/Prague). C18 SepPak cartridges were purchased from Waters Corp., , and ammonium hydroxide (30%) (J.T. Baker brand) was obtained from Avantor Performance Materials .Chemicals were obtained from Aldrich Chemical Company  and were used without further purification unless otherwise stated. Unmodified nucleoside phosphoramidites and 2-dimethoxytrityloxymethyl-6--hexyl-1-O-[(2-cyanoethyl)-(v/v) was used as a matrix.Negative ion MALDI mass spectra were recorded on an Axima Performance  instrument equipped with a nitrogen laser (337 nm) in the linear mode. A mixture of a 50 mg/mL solution of 3-hydroxypicolinic acid in 50% acetonitrile and a 50 mg/mL solution of diammonium hydrogen citrate in deionized water  were recorded on a Synapt G2 Si high-resolution mass spectrometer (Waters) equipped with a quadrupole-time-of-flight mass analyzer . Raw data were collected in the continuum resolution mode and deconvoluted using the MaxEnt1 algorithm to a zero-charge state mass.All UV absorption measurements were performed in 1 cm path length cells using a GBC Cintra 4040 UV-VIS spectrophotometer . Solutions of the compounds for UV experiments were prepared by dissolving each compound in deionized water, and the measurements were performed at ambient temperature.1, 2, FL-1 and FL-2 was performed according to a previously described procedure [closo-dodecaborane and commercially available nucleoside phosphoramidites. Synthesis at a 0.1 µmol scale was performed on an H6 GeneWorld automated DNA/RNA synthesizer  under the conditions recommended by the manufacturer. After synthesis, 6-FAM-labeled oligonucleotides and tripeds 1 and 2 bearing 5′-DMTr protecting groups were cleaved from the solid support under standard conditions. All the compounds were purified by reverse-phase HPLC and desalted on C18 SepPak cartridges. In the case of tripeds 1 and 2, subsequent removal of the 5′-DMTr groups was performed on C18 SepPak cartridges by treating the adsorbed oligomers with 2% TFA, followed by washing the cartridges with water (approximately 10 mL) and eluting the desired product with 30–50% CH3CN in water (approximately 2.0 mL). The resulting tripeds 1, 2, FL-1, and FL-2 were analyzed by ESI-Q-TOF MS and UV-VIS spectroscopy  and FL-2 (RT = 13.7 min). UV detection was performed at λmax 260 nm and λmax 494 nm, and the amount of compound was defined in optical units (OD) at the 260 nm wavelength.Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses were performed on a Shimadzu Prominence HPLC system  using a Kinetex 5 µm C-18 column . All analyses were performed at ambient temperature. The HPLC conditions were as follows: buffer A: 0.1 M CH1, 0.12 OD of triped 2, and 0.1 OD of 22-nt RNA or ASO-22 were dissolved in 15 µL of Milli-Q water and mixed with 32P-γ-radiolabeled ATP , T4 polynucleotide kinase , and kinase buffer (2 µL) supplied by the manufacturer. The reaction mixtures, which had a total volume of 20 µL, were incubated at 37 °C for 1 h. Next, an enzyme was inactivated by incubation of the reaction mixtures for 3 min at 80 °C and the 5′-radiolabeled products were used in studies without further purification.In the experiment, 0.10 OD of triped 1 or triped FL-1 (0.10 OD) with triped 2 or triped FL-2 (0.12 OD), respectively, at a molar ratio of 1:1 in 20 mM Tris-HCl buffer (pH 8) containing 50 mM NaCl and 10 mM MgCl2 in a total volume of 10 µL. The mixture was heated for 5 min at 75 °C, slowly cooled (3 h) to room temperature, and then left overnight at 4 °C. The samples were analyzed by nondenaturing (no urea added) 15% polyacrylamide gel electrophoresis (PAGE). A dsDNA marker  was used in a total volume of 20 µL per lane . For 32P-labeled 1/2, the gels were at first radioautographed, and then stained with Stains-All.The formation of DNA nanostructures was initiated by mixing triped per lane . Electro2. The A431 (human squamous carcinoma) and MCF-7 (human breast cancer) cell lines  were cultured in DMEM containing 4.5 g/L D-glucose, 0.11 g/L sodium pyruvate, and without L-glutamine  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/mL penicillin, and 100 µg/mL streptomycin  at 37 °C and 5% CO2. Cell culture was analyzed for mycoplasma contamination with the EZ-PCR Mycoplasma Detection Kit  and proved negative  cells were cultured in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/mL penicillin, and 100 µg/mL streptomycin  at 37 °C and 5% COnegative . Immedia2+, Ca2+) and lysed in 100 µL of RIPA buffer  with 1 µL protease inhibitor (100×). After 30 min of incubation on ice, the samples were centrifuged at 14,000 rpm for 15 min at 4 °C, and the protein pellet was collected.A431 cells were cultured as described above. The cells were trypsinized , no phenol red) and counted. The cells were washed with 100 µL of cold PBS  and triped 2  were mixed at a molar ratio (1:1) in 20 mM Tris-HCl buffer (pH 8) containing 50 mM NaCl and 10 mM MgCl2 in a total volume of 15 µL. The mixture was heated for 5 min at 75 °C, slowly cooled (3 h) to room temperature, and left overnight at 4 °C. Oligonucleotide ASO-22 (4 µL of stock solution after phosphorylation) was mixed with 20 mM of Tris-HCl buffer (pH 8) containing 50 mM NaCl and 10 mM MgCl2 in a total volume of 15 µL. The samples of 1/2 (15 µL) or ASO-22 (15 µL) were mixed with A431 cell lysate . The mixture was incubated at 37 °C for up to 48 h. Aliquots of the enzymatic reaction mixture (4 µL) were withdrawn from the reaction mixture at predetermined times  and mixed with loading buffer  (6 µL). Each sample was analyzed by nondenaturing (no urea added) 15% PAGE at room temperature at 20 mA for 2 h. Gels were visualized on radiographic films (1/2 and ASO-22 decay courses (The  courses D were as32P-radiolabeled substrate RNA (2 µL of the stock solution after phosphorylation) was mixed with triped 1 or ASO-22  in 20 mM Tris-HCl buffer (pH 8) containing 50 mM NaCl and 10 mM MgCl2 in a total volume of 7 µL. The mixture was heated for 5 min at 75 °C and slowly cooled (1 h) to room temperature. To each sample, 10x reaction buffer containing 200 mM Tris-HCl, 500 mM KCl, 200 mM DTT, and 50 mM MgCl2 (3 µL) and Milli-Q water (19 µL) were added at 4 °C, and then ribonuclease H  (1 µL of 1 U/µL stock solution) was added to a total volume of 30 µL. The resultant assay mixture was incubated at 37 °C for up to 90 min. Aliquots of the enzymatic reaction (3 µL) were withdrawn from the reaction mixture at predetermined times  and mixed with loading buffer  (6 µL), and the enzyme was inactivated by incubation of the reaction sample for 3 min at 80 °C. Each sample was analyzed by 20% PAGE with 7 M urea at room temperature at 20 mA for 2.5 h. After electrophoresis was complete, the gels were transferred to an exposure cassette and covered with autoradiography double-coated films  for 10 min at low temperature (−25 °C). Then, the double-coated films were soaked in the developing reagent  and then in the fixing reagent and scanned using a G-Box apparatus . The mixture of 32P-isotope labeled oligoribonucleotides (2–22 mers) synthesized in house was used as a marker (M) for analysis of data in In the experiment, 3 cells per well in 100 µL of complete medium and cultured for 24 h to reach 80% confluence. Immediately before transfection, the cell medium containing antibiotics was replaced with fresh medium without antibiotics. After 1 h of incubation, A431 cells were transfected with complexes 1/2 or ASO-22 dissolved in 50 µL OPTI-MEM medium at concentrations of 0, 5, 25, 50, and 100 nM using Lipofectamine 2000 at a ratio of 2:1 (2 µL of Lipofectamine 2000 per 1 µg of nucleic acid). For control the cells were Lipofectamine 2000-transfected with ASO-C (100 nM). After 48 h of incubation at 37 °C in a 5% CO2 atmosphere, cells were washed with cold PBS and lysed with 99 µL of RIPA buffer and with 1% protease inhibitor 100x (1 µL). Samples were incubated for 30 min at 4 °C and centrifuged at 14,000 rpm for 15 min at 4 °C. The protein concentration was determined by the Bradford protein assay , and 30 µg protein per well was separated on SDS PAGE (4% and 8%) reducing gradient gels  and transferred to nitrocellulose membranes  for semi-dry Western blot analysis. After blocking nonspecific binding sites, membranes were incubated overnight at 4 °C with primary antibodies: anti-actin Mab (1:500) mouse monoclonal antibody, clone C4 , and anti-EGFR A10 (1:200) mouse monoclonal antibody .A431 cells were cultured as described above. Before transfection, the medium was removed, and cells were trypsinized , no phenol red) and counted. Then, cells were loaded in 96-well plates with black walls and a transparent bottom (PerkinElmer) at a density of 15 × 10Membranes were washed 3 times for 20 min at room temperature before incubation with a secondary antibody for 1 h at 4 °C. The goat anti-mouse IgG (1:5000) (H&L) peroxidase  was the secondary antibody. Membranes were scanned using ELC Western Blot Pierce Blotting Substrate  for 5 min in darkness and analyzed using a G-Box apparatus . Experiments were conducted in duplicate and the mean with standard deviation (±SD) is given.1/2 or ASO-22 (2 µL of Lipofectamine 2000 per 1 µg of the nucleic acid) used at concentrations of 0, 50, and 100 nM dissolved in 50 µL OPTI-MEM medium. For control the cells were Lipofectamine 2000-transfected with ASO-C (100 nM). For the wound-healing assays, after 5 h of transfection at 37 °C in an atmosphere of 5% CO2, cells in 96-well plates were wounded by scraping with pipette tips (of 20 µL size). The cells were washed two times with a medium, and the wound areas were marked and photographed at different points using a digital camera attached to a phase-contrast microscope . The number of cells in the wound region was counted. Experiments were conducted in triplicate and the mean with standard deviation (±SD) are given.HeLa cells were cultured and transfected as described above. After 1 h of incubation, HeLa cells were transfected with Lipofectamine 2000 at a ratio of 2:1 with After 48 h of incubation, HeLa cells in each well were photographed at the same points for phenotypic change assays and wound healing assays. Cells in the wound areas were counted under a bright-field microscope at a magnification of 200× across 4 randomly selected fields.1/2 or ASO-22 as described above. A431 cells treated with Lipofectamine 2000 at the concentration corresponding to transfection of 1/2 at 100 nM were used as a control. After 48 h of incubation at 37 °C in an atmosphere of 5% CO2, under the same conditions, cells were harvested and transferred to centrifuge tubes and spun for 5 min at 1100 rpm. Each cell sample was then suspended in cold 70% ethanol for fixation for 1 h. Then, the cells were centrifuged for 10 min at 1500 rpm and 4 °C and washed twice in PBS without Ca2+ and Mg2+ ions. Next, cells were treated with 10 µL of RNase A (10 mg/mL) for 30 min at 36 °C to ensure that only DNA, and not RNA, was stained. In the next step, 5 µL of propidium iodide (1 mg/mL) was added, and the cells were incubated at room temperature for 30 min. Each cell sample was then transferred to a 5 mL polystyrene round-bottom tube . Flow cytometry and corresponding fluorescence measurements were performed using a BD FACSCalibur flow cytometer  using the FL2 Red channel. The number of events was stopped at 25,000 counts. Data collected from the experiments were analyzed using ModFit LT software. FACS analysis with confidence interval (CI) and the mean with standard deviation (±SD) statistics is given in A431 cells were cultured and transfected with nanostructures 2+ and Mg2+) and imaged under a fluorescence microscope  to detect DAPI at λex = 340–380 nm  and FITC at λex = 465–495 nm , B-2A , TX red , and G-2A longpass λex = 510–560 nm , where DM is a dichroic mirror and BA is an emission filter.MCF-7, HeLa, and A431 cells were cultured and transfected as described above. After 5 h of incubation, the transfection mixture was replaced with a full fresh medium with antibiotics in a volume of 200 µL per well. Next, after 6 h of incubation, the full medium was mixed with 5 µg/mL DAPI dye (which stains cell nuclei blue)  and 1 µmol/mL ER-Tracker Red dye (which stains ER membranes red) . The cells were incubated for 30 min with the dyes in the dark at 37 °C. After incubation, the cells were washed two times with PBS buffer (with Ca1/2 or ASO-22 (50 nM) for 16 h or with control nigericin  for 4 h to activate the NLRP3 inflammasome. The level of the released IL-1β was measured from conditioned macrophage media by enzyme-linked immunosorbent assay  according to the manufacturer’s instructions. Experiments were conducted in two independent donors.Peripheral blood mononuclear cells were isolated from leukocyte-rich buffy coats provided commercially by blood transfusion and donation center . The blood donors agreed on donating the buffy coat randomly for research purposes during blood donation, maintaining their anonymity. Monocytes selected by adhesion to plastic were differentiated into macrophages in macrophage-SFM medium  supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor  and antibiotics as described previously ,72. On d1/2 and ASO-22 (50 nM) in macrophages was assessed with the use of the MTT assay. The macrophages were cultured as described above. The cells were incubated for 16 h with tested compounds at 37 °C under a 5% CO2 atmosphere, followed by the addition of the MTT solution in PBS (5 mg/mL) to each well. The cells were then incubated for 3 h at 37 °C under a 5% CO2 atmosphere. Finally, 95 µL of lysis buffer  was added to each well and cells were incubated overnight at 37 °C. The sample absorbance was measured at two wavelengths: 570 nm and the reference wavelength of 630 nm . The percentage of living cells (%LC) was calculated from the equation: %LC = Asample × 100%/(Acontrol cells) where Asample is the absorbance of a given sample of cells treated/transfected with the tested compound and Acontrol cells is the absorbance of the untreated cells (100% viability). The results are mean values ±SD from two technical experiments.The cytotoxicity of boron clusters  as well as 1/2  and Lipofectamine 2000 at a ratio of 1:2 dissolved in 50 µL OPTI-MEM medium. After 5 h of incubation, the transfection mixture was replaced with a fresh medium containing antibiotics in 200 µL per well and the cells were incubated for 48 h at 37 °C in an atmosphere of 5% CO2. In order to conduct the experiment for the assessment of the free uptake of 1/2, the medium was replaced to basic medium  , 100 U/mL penicillin, and 100 µg/mL streptomycin  and the aqueous solutions of 1/2  were added to the medium and the cells were incubated for 48 h at 37 °C in an atmosphere of 5% CO2.A431 cells were cultured as described above and then transfected with aqueous solutions of After 48 h (experiments with or without Lipofectamine 2000) and medium removal, the cells were treated with trypsin  and diluted with ultrapure water  up to a 1.5 mL volume; then, the ICP MS measurements were performed and data were collected. The boron content was determined with a Varian 810-MS inductively coupled plasma mass spectrometer  equipped with a Micromist nebulizer, quartz Scott spray chamber (3 °C), platinum sampler cone, and nickel skimmer cone. The operating parameters were as follows: RF power: 1.4 kW; plasma flow (argon): 17 L/min; auxiliary flow (argon): 1.7 L/min; nebulizer flow (argon): 1.00 L/min; pump rate: 4 rpm; sheath gas (argon): 0.2 L/min; and number of scans: 10 m/z: 10 and 11.10B and 11B). Six independent experiments were performed.The calibration curve method was applied. A series of 10 calibration solutions were prepared in the boron concentration range of 0.1–50 µg/L by appropriate dilution of the standard solution of boron  using ultrapure water. A linear model of the calibration curve was adopted, with a minimum correlation coefficient of 0.999. The obtained results were the average of concentrations calculated from the calibration curves obtained for both stable boron isotopes  or 2′-OMe at their 5′-ends. Moreover, the attachment of additional boron clusters and their enrichment with 10B isotope atoms may allow the 1/2 nanocarriers to achieve a 10B concentration sufficient for successful BNCT. Thus, the favorable properties of 1/2 nanostructures, both in terms of their proven antisense properties and their potency to serve as carriers of boron atoms for BNCT, make these constructs of interest for further development as anticancer agents.Nanostructures built of tripeds


Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The deamination is usually performed using sodium bisulfite solutions of 3–5 M concentration with an incubation period of 12–16 hr at 50 °C. We demonstrate here that this deamination can be speeded up significantly. We prepared a solution of 10 M bisulfite concentration of pH 5.4 and used it to treat DNA at temperatures up to 90 °C. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at 90 °C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer a significantly improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation. Bisulfite-mediated deamination of cytosine is a chemical reaction discovered in 1970.5)In studies on the functional consequences of methylation in DNA, ‘bisulfite genomic sequencing’ is a principal methodology for determining the methylated sites.Cytosine, either as a base, a nucleoside, or a constituent of polynucleotides, can be converted into its 5,6-dihydro-type adduct [1] when mixed with bisulfite in aqueous solutions .Sodium bisulfite, sodium sulfite (Reagent Grade), ammonium sulfite monohydrate, 50% ammonium bisulfite solution, and 0.1 N HCl were purchased from Wako . Nucleosides and salmon testis DNA were products of Sigma . Plasmid pUC119 was obtained from TaKaRa . Restriction endonuclease Reactions and manipulations at desired temperatures were performed using, unless otherwise noted, thermostatted water baths with an accuracy of ± 0.1 °C . Solubilization of reagents in aqueous media was done by warming the mixture at a desired temperature for 5–10 min with frequent vigorous shaking. For spectrophotometry, a Hitachi model U-2800 UV-Vis spectrophotometer  was used.276 was measured. 1 M Na2SO3 was used as a standard: the A276 for this standard showed a linear dose-response at 0.5 mM-3 mM (A276 0.2–1.2).To assay concentrations of bisulfite in solutions, we devised a spectrophotometric method based on the absorbance of sulfur dioxide in acidic solutions, i.e., absorbance at 276 nm in 0.1 N HCl .270 was determined. Decrease in A270 from an appropriately prepared untreated-sample  is the measure of deamination ). With 5-methyl-2′-deoxycytidine, A277 was employed in place of A270 for the rate measurement.For reaction rate measurements, the deamination extents were determined spectrophotometrically as described previously.3 and Na2SO3  into 50% ammonium bisulfite solution to make a final volume of 10 ml. The bisulfite content found for the prepared solutions was 7 M.To determine the pH dependence of the rate, bisulfite solutions of various pH values were prepared by dissolving mixtures of NaHSOThe analysis was performed using a Hitachi L-7100 pump , a Beckman-Coulter column Ultrasphere ODS 4.6 mm × 25 cm , and a 260 nm UV-detector. Ten μl of sample was injected, and eluted with a linear gradient composed of buffers A and B  at a flow rate of 0.7 ml/min using 100 mM K-phosphate, pH 7, as buffer A, and 90% methanol-1 mM K-phosphate, pH 7, as buffer B. The retention time of standard nucleosides in this HPLC was 19 min for 2′-deoxycytidine, 22 min for 2′-deoxyuridine, 25 min for 5-methyl-2′-deoxycytidine, 26 min for 2′-deoxyguanosine, 28 min for thymidine, and 32 min for 2′-deoxyadenosine.4)HSO3-NaHSO3 solution, pH 5.4. The mixture was incubated using a TaKaRa PCR thermal cycler, first at 95 °C for 30 sec and then at a desired temperature. Upon completion of the treatment, the sample containing 80 μg of DNA in 605 μl in volume was applied onto Sephadex G-50 column  equilibrated with 10 mM Tris-HCl-1 mM EDTA, pH 8, and eluted with the same buffer to obtain a DNA fraction. DNA was collected by precipitation with ethanol and was dissolved in 100 μl of H2O. To generate uracil from uracil bisulfite adducts, 90 μl of the sample solution was added to 11 μl of 2 N NaOH, and incubated for 10 min. DNA was collected by ethanol precipitation, digested with DNase I (Boeringer), snake venom phosphodiesterase (Worthington) and calf intestinal alkaline phosphatase (Promega) into nucleosides, and submitted to HPLC analysis.Salmon testis DNA denatured in 0.3 N NaOH for 30 min at 30 °C was mixed with 10 volumes of 10 M  following the manufacturer’s recommendation and finally dissolved in 90 μl of water. Eleven μl of 2 N NaOH was added to the DNA solution and the resulting mixture was incubated at 37 °C for 10 min. DNA was recovered by ethanol precipitation, with yeast tRNA  added as a carrier, and was dissolved in 100 μl of TE .One μg of One μl of this DNA solution was subjected to the PCR reaction. AmpliTaq DNA polymerase Stoffel fragment  was used following the instructions of the manufacturer. After a 3-min incubation at 95 °C, 30 cycles of amplification  was performed. The PCR primers used were 5′-CGGAATTCTATTGGTTAAAAAATGAG-3′ and 5′-AACTGCAGACATTAACCTATAAAAATA-3′. The reaction volume was 50 μl. One μl of this PCR product was subjected to agarose gel electrophoresis.2SO3 as a standard for determining bisulfite content of a given reagent, we observed that the NaHSO3  contained 1.11 mol of bisulfite per mol formula-weight , and that the (NH4)2SO3≥H2O contained 0.92 mol bisulfite per mol formula-weight. 50% Ammonium bisulfite solutions (3 different bottles tested) were found to contain 6.0–6.2 M bisulfite.By using Na3 can be dissolved up to 5.9 M at 70 °C, and 5.0 M at 30 °C; Na2SO3 to 2.1 M at 70 °C, and 1.5 M at 30 °C; and (NH4)2SO3≥H2O to 4.3 M at 70 °C and 3.5 M at 30 °C. The highest bisulfite concentration obtainable by dissolving a mixture of NaHSO3 and Na2SO3 at 70 °C to give a solution of pH 5.4 was 5.9 M.First, we determined the highest bisulfite concentrations obtainable by dissolving sodium bisulfite, sodium sulfite or ammonium sulfite in water. Thus, NaHSO3 and 0.67 g (NH4)2SO3≥H2O in 5.0 ml 50% (NH4)HSO3 (pH 4.5) by heating at 70 °C for 5–10 min, a solution of pH 5.4 containing 10 M bisulfite was obtained . This solution is stable: thus, when a freshly prepared sample was allowed to stand in a tightly stoppered container at 70 °C, the bisulfite content and the pH value showed no change after 4 hr.Next, the availability of 50% ammonium bisulfite solution from a commercial source enabled us to prepare solutions of higher bisulfite concentrations. By dissolving a mixture of 2.08 g NaHSO1/2) of deoxycytidine at 70 °C in 5.3 M NaHSO3-Na2SO3 was 3.0 min, while that in 9 M (NH4)HSO3-NaHSO3 was 1.8 min. Thus, the rate of deamination (1/t1/2) was indeed proportional to the bisulfite concentration: [t1/2 at 5.3 M]/[t1/2 at 9 M], 1.7, was equal to [9 M]/[5.3 M]. This proportionality was also observed for reactions conducted in bisulfite solutions stepwise-diluted from 9 M bisulfite (data not shown). The half lives of deoxycytidine in 9 M bisulfite, pH 5.4, at temperatures other than 70 °C were as follows : less than 1 min/90 °C, 5 min/50 °C, and 17 min/37 °C. Deamination of 5-methyl-2′-deoxycytidine in 9 M (NH4)HSO3-NaHSO3 was slow both at 70 °C (16% at 10 min) and at 90 °C (23% at 10 min)(t 10 min).Quantitative conversion of 2′-deoxycytidine into 2′-deoxyuridine in these reactions was confirmed by HPLC analysis. Thus, after the bisulfite treatment, the mixture was treated with alkali, and subjected to HPLC analysis. We observed 100% conversion of 2′-deoxycytidine into 2′-deoxyuridine for reactions in 9 M bisulfite at pH 5.4, both in a reaction at 90 °C for 8 min and a reaction at 70 °C for 30 min.The pH dependence of the deamination was determined using 50% ammonium bisulfite containing 1 M sodium bisulfite at pH 4–6. The temperature employed in this experiment was 60 °C, at which the reactions are slower than at 70 °C to allow more accurate determination of the reaction extents. As Hydroquinone, which has been used previously for the prevention of bisulfite decay,Salmon testis DNA was treated with 9 M bisulfite. As shown in For this efficient deamination to be useful in genomic DNA sequencing, it is required that the bisulfite-treated DNA can be amplified by PCR. We treated a cloned DNA fragment with the 9 M bisulfite at 90 °C for 10 min, the DNA was recovered, and then investigated whether the treated DNA fragment can serve as a template for PCR amplification. Amplified fragments were detected in approximately equal amounts for DNA samples that had undergone treatments at 90 °C or at 70 °C for periods of 5, 10, 20 and 40 min (data not shown). This observation indicates that the DNA did not undergo serious degradation during these treatments.We expect that this new deamination procedure would be useful in speeding up the analysis of methylated sites in DNA. Such a significant improvement in the bisulfite genomic sequencing will greatly promote research on the biological and medical consequences of DNA methylation.