These nanosheets are epitaxially deposited on surfaces of semiconducting SnS2 nanoplates to form vertical heterostructures. The ohmic-like contact formed at the Sn0.5W0.5S2/SnS2 heterointerface affords rapid transport of charge carriers, and allows for the fabrication of fast photodetectors. Such facile charge transfer, combined with a high surface affinity for acetone molecules, further enables their use as highly selective 100 ppb level acetone sensors. Our work suggests that combining compositional and structural control in solution-phase epitaxy holds promises for solution-processible thin-film optoelectronics and sensors.The creation of crystal phase heterostructures of transition metal chalcogenides, e.g., the 1T/2H heterostructures, has led to the formation of metal/semiconductor junctions with low potential barriers. Very differently, post-transition metal chalcogenides are semiconductors regardless of their phases. Herein, we report, based on experimental and simulation results, that alloying between 1T-SnS 0.5W0.5S2 nanosheets with 83% metallic phase on SnS2 nanoplates, which are used as 100 ppb level chemiresistive gas sensors at room temperature.Controlling the composition and crystal phase of layered heterostructures is important. Here, the authors report the liquid-phase epitaxial growth of Sn As a group of the mostly studied layered materials, transition metal chalcogenides (TMCs) like MoS2 and WS2 exist in different crystal phases such as the 2H and 1T polytypes with distinct electronic properties. Their semiconductor-to-metal transition  can be realized via Li-intercalation,7 strain engineering,8 e-beam/laser irradiation2 or doping/alloying9. This attractive feature allows for the formation of 2H/1T  phase junctions that exhibit much reduced contact resistance compared to that of using noble metal contacts  which normally interface with semiconducting TMCs with large Fermi level misalignment10. Besides TMCs, post-TMCs such as SnS2 and InSe are another important group of layered materials, exhibiting attractive electronic and optoelectronic properties for a wide range of applications, including transistors, photodetectors, and sensors12. Similar to many other semiconducting materials, the type of metal contact with post-TMCs plays a critical role in tuning their functional performance13. However, due to the less metallic nature of post-transition metals, post-TMCs are generally semiconductors regardless of their crystal phases, such as the 2H, 4H, and 1T polytypes14. Consequently, contacting post-TMCs with metallic layered materials to achieve low interfacial resistance remains a big challenge.Heterostructures constructed from layered materials such as graphene, metal chalcogenides and black phosphorus (BP) have aroused particular interest due to their combined advantageous features and the emergence of unusual properties/functions15. In particular, to prepare vertical heterostructures in which dissimilar layered crystals are stacked one above the other in a pre-designed sequence, solid-state procedures, including dry transfer, chemical vapor deposition (CVD) and chemical vapor transport (CVT) method have mostly been applied17. This is because these methods allow good control over the spatial arrangement of the layers as well as the deposition sequence. Compared to these solid-state methods, solution-phased approaches are advantageous in terms of relatively easier procedures, low-cost setups, and most importantly, scalability19. However, direct wet-chemical growth of vertical heterostructures of layered metal chalcogenides has thus far been challenging.To date, much effort has been devoted to the preparation of various heterostructures based on layered materials, which display different geometric arrangements such as the lateral and vertical heterostructures2/MoS2, MoS2/perovskite, and graphene/MoS2/graphene have recently shown promising performance in photodetectors, due to the improved charge separation/transport and enhanced light adsorption23. Meanwhile, development of gas sensors for detection of volatile organic compounds are important in applications such as environmental monitoring and non-invasive diagnosis of diseases based on breath analysis25. Chemiresistive sensors based on metal oxides/sulfides have been used for detection of volatile organic compounds, however, high operating temperatures  are usually required to achieve good sensitivity and selectivity26. Very recently, layered materials such as SnS2, WS2 and Ti3C2Tx have demonstrated great potential for room-temperature gas detection29. It is expected that creation of heterostructures may realize further improved sensing performance32.In view of their potential applications, heterostructures/heterojunctions such as InSe/graphene, MoTe2, a typical n-type semiconductor, are used as synthesis templates for the surface deposition of layered Sn0.5W0.5S2 nanosheets, which show 83% metallic phase, leading to the formation of metal/semiconductor vertical heterostructures. Kelvin probe force microscope (KPFM) and tunneling atomic force microscopy (TUNA) analyses suggest the formation of ohmic-like contact at the Sn0.5W0.5S2/SnS2 interface. The resultant heterostructures are fabricated into chemiresistive sensors to detect acetone at room temperature and exhibit a good selectivity and a minimum detectable concentration down to 100 ppb. The good sensing performance could be attributed to the low charge transfer resistance at the Sn0.5W0.5S2/SnS2 interface that enables a much increased signal-to-noise ratio, and the alloying induced enhancement in surface gas adsorption.In this contribution, nanoplates of SnS2 has been widely studied and applied in phototransistors and sensors for its favorable band structure and relatively high surface electronegativity33. Typically, SnS2 nanoplates were synthesized via a hydrothermal reaction with thiourea (CS(NH2)2) and tin tetrachloride hydrate (SnCl4•5H2O) as the precursors for S and Sn, respectively (see the Methods section for the detailed procedure)34. As shown in the scanning electron microscope (SEM), transmission electron microscope (TEM) and atomic force microscopy (AFM) images in Supplementary Figs. 2 with a 1T structure (space group Pm1), where a = 3.65 and c = 5.90 (ICSD no. 42566)12 10H2(W2O7)6 to the precursors of the aforementioned synthesis solution, alloyed Snx1–WxS2 nanosheets were in-situ synthesized and hybridized with SnS2 nanoplates as shown in Fig. 2 nanoplate covered by Snx1–WxS2 nanosheets on both its basal faces forming a vertical heterostructure. The deposited Sn1–xWxS2 nanosheets are typically 6–9 nm in thickness  and (002) planes of Sn0.5W0.5S2 nanosheets with an enlarged interlayer spacing of about 1.0 nm. The enlarged spacing, which was also observed in side-view HRTEM images 2 and (NH4)10H2(W2O7)6 in our synthesis, leading to the intercalation of NH4+ ions in between the adjacent layers35. This was also confirmed by X-ray photoelectron spectroscopy (XPS) analysis  lattice spacing is 3.16 Å, in good agreement with the XRD result  planes was 3.0 Å, corresponding to a lattice parameter of a = 3.46 Å. This reasonably falls in between that of WS2 (a = 3.16 Å) and SnS2 (a = 3.65 Å). Note that the mismatch between the (100) planes of SnS2 and Sn0.5W0.5S2 is 5%, which can be tolerated in van der Waals epitaxial growth of layered materials36. The top-view HRTEM image in Fig. 2 nanoplate covered with Sn0.5W0.5S2, and the brighter region for the periphery Sn0.5W0.5S2 nanosheet. The lattice fringes extend continuously from the SnS2/Sn0.5W0.5S2 center to the Sn0.5W0.5S2 edge, further confirming the epitaxial growth mode. Moiré patterns could be observed in some areas due to the overlap of Sn0.5W0.5S2 and SnS2 at small misorientation angles. For example, as shown in Fig. 0.5W0.5S2 overlayer made a misorientation angle of 3° with SnS2  images of the edge area of a heterostructure show the 1T-phase-like atomic arrangement of Sn0.5W0.5S2  calculation results shown in Supplementary Table −1 that correspond to the SnS2-like A1g, WS2-like E2g and WS2-like A1g modes, respectively, the active modes observed at 171 cm−1 and 224 cm−1 in the lower frequency region could be attributed to distorted 1T-WS235. The dominant distorted 1T-phased W–S coordination was further confirmed by XPS analysis  associated with W in the 1T-phased structure are downshifted by 0.6 eV relative to those associated with W in the 2H structure (32.7 and 34.8 eV). The deconvolution of the W 4f bands thus could enable the quantitative estimation of the 1T and 2H phase concentrations7. In our case, from the deconvoluted peak areas  analysis. In the XRD pattern in Fig. ult Fig. . The othsis Fig. . It has eas Fig. , the conure Fig. 44.Fig. 2+, K+ etc.)45 or synthesized in the presence of ammonium containing precursors35 and hydrazine hydrate46. According to previous theoretical calculations, the presence of the positive counterions could cause an increase of the electron density of the d-orbital of the transition metals, leading to the stabilization of the 1T or distorted 1T phase47. Therefore, the realization of the distorted 1T-Sn0.5W0.5S2 in our present work might also be a result of the intercalated NH4+ cations from CS(NH2)2 and (NH4)10H2(W2O7)6 used in the synthesis solution.The formation of 1T or distorted 1T structures have been observed previously in TMCs when they were intercalated with alkali metal ions 10H2(W2O7)6 and SnCl4·5H2O, reacted to produce Sn(HWO4)2•nH2O amorphous particles upon mixing at 80 °C  are shown in Fig. 2 which shows no DOS at the Fermi level  also shows the metallic behavior  vs. drain-source voltage (Vds) curves at varied gate voltages reveal that the SnS2 nanoplates are typical n-type semiconductors  of 5.100 eV was used as the potential reference. The 2D potential image of a typical Sn0.5W0.5S2/SnS2 heterostructure is shown in Fig. V = ɸAu − ɸsample) . It can be seen that the surface potential of the Sn0.5W0.5S2/SnS2 heterostructure is 0.029 V lower than that of Au to give an estimated work function of 5.071 eV  with TUNA , which is comparable and outperforms some previously reported TMC based photodetectors59. This value is also about 50 times shorter than that of the SnS2-based photodetector (2.10 s)  at room temperature  with increasing acetone concentration  at 100 ppb levels among all reported chemiresistive sensors operating at room temperature , indicating its good repeatability.The presence of a metallic component in a thin film channel may pose some limitation to its optoelectronic performance, such as the relatively large dark current observed in the aforementioned Snure Fig.  and 21. ion Fig. . A minim0.5W0.5S2 nanosheets on SnS2 nanoplates could be attributed to the following reasons. First, the formation of an ohmic-like contact between Sn0.5W0.5S2 and SnS2 that allowed rapid charge transfer across the metal–semiconductor interface partly led to a 35 times reduction in background noise and thus a much higher signal to noise (S/N) ratio . This indicates that acetone molecules interact more strongly with Sn0.5W0.5S2. It is worth noting that the NH4+-intercalation did not significantly change the adsorption ability of Sn0.5W0.5S2 nanosheets towards acetone, with the adsorption energy slightly reduced by 0.09 eV. As further shown in Fig. 0.5W0.5S2 due to charge transfer from the absorbed acetone molecule. Third, one of the advantages of using low-dimensional materials in gas sensing as compared with bulk crystals is their large specific surface areas, which are beneficial for providing large interfaces for channel–gas interaction25. Indeed, the deposition of wrinkled Sn0.5W0.5S2 nanosheets on SnS2 nanoplates increased the specific surface areas from typically 6.25 to 11.37 m²/g based on the Brunauer–Emmett–Teller (BET) measurements as shown in Supplementary Fig. 0.5W0.5S2 nanosheets and their strong interaction with acetone, increasing the amount of Sn0.5W0.5S2 nanosheets on SnS2 did not further improve the sensing performance, but on the contrary, led to poorer sensitivity with a minimal detectable concentration of only 1 ppm  was identified as an intermediate product which formed at 180 °C and decomposed subsequently at 220 °C to provide additional Sn and W ions with a high W/Sn ratio of about 30. We proposed that this high W/Sn concentration could drive the formation of alloyed Sn0.5W0.5S2 nanosheets despite the fact that the formation of Sn–S bond requires less energy compared to that of the W–S bond. Although 1T-SnS2 is a semiconductor, alloyed Sn0.5W0.5S2 showed 83% distorted 1T structure, which is metal-like as predicted by theoretical calculations. KPFM and TUNA measurements on a Sn0.5W0.5S2/SnS2 heterostructure suggested the formation of ohmic-like contact at the heterointerface, resulting in a low charge transfer resistance. The rapid charge transport at the Sn0.5W0.5S2/SnS2 heterointerface allowed for the fabrication of fast photodetector with a short response time of 42 ms. Additionally, when the Sn0.5W0.5S2/SnS2 heterostructures were fabricated into thin films for gas sensing, a much enhanced signal-to-noise ratio was achieved partly due to the presence of metallic Sn0.5W0.5S2 layers. Furthermore, Sn0.5W0.5S2 showed much enhanced surface adsorption of acetone than SnS2 based on theoretical calculations. As a consequence, selective and sensitive detection of acetone was achieved with an ultralow minimum detectable concentration of 100 ppb at room temperature. This, combined with large sensing responses at 100 ppb levels outperforms previously reported room-temperature chemiresistive sensors. Our use of semiconducting SnS2 nanoplates as synthesis templates for the solution-phase epitaxial growth of metallic Sn0.5W0.5S2 nanosheets demonstrates a promising way towards the facile, economic and high-yield preparation of functional hybrid nanomaterials. Synthesis of layered materials through alloying among elements with distinct electronic and chemical properties is expected to bring about more materials and unusual phenomena.The epitaxial growth of metallic Sn4)10H2(W2O7)6, 99.99%), ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O, 99.0%), thiourea (CS(NH2)2, 99.0%) and tin tetrachloride hydrate  were purchased from Sigma-Aldrich . Ethanol  was purchased from J&K chemical . The gaseous analytes 2O, CH3CH2CHO, C6H5CH3, and NO2) which were diluted with N2 gas at concentrations of 1000 ppm were purchased from Nanjing Teqi Co., Ltd. All chemicals were used as received without further purification. The deionized (DI) water was purified using Milli-Q3 System .Ammonium tungstate hydrate ((NH4·5H2O and 3.75 mmol of CS(NH2)2 were dissolved in 19.45 mL DI water and stirred for 2 h to form a homogeneous solution. This solution was transferred to a 25 mL Teflon-lined stainless steel autoclave, heated to 220 °C in an electrical oven and then maintained at this temperature for 12 h before being cooled down naturally to room temperature. The obtained product was centrifuged at 8000 rpm for 10 min, and the precipitate was washed with DI water for three times before further characterization.In a typical process, 0.25 mmol of SnCl4)10H2(W2O7)6, 7.5 mmol of CS(NH2)2, and 0.5–0.625 mmol of SnCl4·5H2O were dissolved in 19.45 mL DI water and stirred at 80 °C for 2 h to form a homogeneous solution. This solution was then transferred to a 25 mL Teflon-lined stainless steel autoclave, heated to 220 °C in an electrical oven and then maintained at this temperature for 60 h before being cooled down naturally to room temperature. The obtained product was centrifuged at 8000 rpm for 10 min, and the precipitate was washed with DI water for three times before further characterization.In a typical process, 0.25 mmol of , transmission electron microscope  and high resolution transmission electron microscope  coupled with energy dispersive X-ray (EDX) spectroscope were used to investigate the compositional, morphological and structural features of the samples. X-ray diffraction  was performed using CuKα radiation (λ = 1.54 Å). X-ray photoelectron spectroscopy  measurements were conducted on the different metal sulfide nanostructures, and the binding energies were corrected for specimen charging effects using the C 1 s level at 284.6 eV as the reference. Raman spectra  of the samples were collected with a 532 nm laser. Semiconductor parameter analyzer  and probe station  were used to investigate the semiconductor properties of the samples. A commercial atomic force microscope  was used to investigate the electrical properties of the individual nanostructures in air. Inductively coupled plasma mass spectrometry  was used to measure the concentration of Sn and W ions in the synthesis solution. Brunauer–Emmett–Teller  measurements were carried out to determine the specific surface area and pore size distribution of various samples.2 (285 nm)/Si substrate via thermal evaporation through a shadow mask, SnS2 nanoplates or Sn0.5W0.5S2/SnS2 heterostructures in water were drop-casted onto the substrate, acting as the channel to connect the drain and source electrodes with a channel length of 11 μm. The semiconducting properties of the channel materials were then characterized using a Keithley 4200 semiconductor characterization system operating at 77 K in vacuum (5 × 10−5 Torr).After Au (50 nm)/Cr (30 nm) drain and source electrodes were deposited onto a SiO2 (285 nm)/Si substrate via thermal evaporation, SnS2 nanoplates (or Sn0.5W0.5S2/SnS2 heterostructures) in water were drop-casted onto the substrate. A KPFM  was then used to characterize the surface potential of the SnS2 nanoplates (or Sn0.5W0.5S2/SnS2 heterostructures) at ambient conditions. The contact potential difference between the tip (PtIr) and the sample surface (VCPD), which is also referred to as the surface potential can be calculated by using the following equations:φt, φs, and φf represent the work functions of the probe tip, the substrate, and the sample film, respectively.After Au (50 nm)/Cr (30 nm) was coated onto a SiOI–V) characteristics of individual SnS2 nanoplates or Sn0.5W0.5S2/SnS2 heterostructures. During the measurement, the PtIr tip was pressed against the sample with a constant force, feedback was switched to contact mode, and the voltage was linearly ramped up and down while the current signal was collected. Analysis of the I–V curves was performed with the Nanoscope Analysis software.PeakForce Tunneling atomic force microscopy  was used to investigate the current–voltage (2 (300 nm)/Si substrate via magnetron sputtering through a shadow mask. After that, 0.5 µL of a concentrated dispersion (6.0 mg/mL) of the Sn0.5W0.5S2/SnS2 heterostructures or SnS2 nanoplates was drop-casted on the electrodes.Au (15 nm in thickness) interdigitated electrodes with a 10 nm spacing were deposited onto a SiOI–V) and the current–time (I–t) curves of the photodetectors were measured on a semiconductor characterization system  in air at room temperature. A 405 nm laser was used for all the measurements. The actual power intensity was measured by a power meter .The current–voltage 2O, CH3CH2CHO, C6H5CH3, and NO2. Typically, a drop of 100 μL aqueous solution containing 10 mM as-prepared SnS2 or Sn0.5W0.5S2/SnS2 was drop-casted onto an Au interdigitated electrode  and then dried in oven at 60 °C. The gas sensing test was performed in an air-tight chamber with electrical feedthroughs at room temperature (25 °C). A constant current was applied to the sensor electrode, and the variation of the sensor resistance was monitored and recorded with the changes in the gas environment using a data acquisition system  with a 20 channel multiplexer . A typical sensing measurement cycle consisted of three sequential steps: (1) a dried N2 flow was introduced into the chamber to record a baseline resistance (R0); (2) a target gas, e.g., acetone, balanced in N2 was introduced, and the concentration increased (0.1–50 ppm) with progressive cycles; and (3) when the resistance of the sensor reached equilibrium in the target gas (Ra), the target gas was replaced by N2 to allow the resistance of the sensor to return to R0. All gas flows were set at 500 sccm, precisely controlled by using mass-flow controllers.Chemiresistive gas sensors were fabricated based on SnS67. The exchange-correlation interaction uses the general gradient approximation (GGA) formulated by Perdew–Burke–Ernzerhof (PBE)66. All electron interactions were described with projector augmented wave (PAW) pseudo potentials. Long-range dispersion corrections have been considered within the DFT-D2 method. The dispersion coefficient C6 and van der Waals radius R0 for H, C, N, O, S, Sn and W used in our DFT-D2 method were taken from previous reports69. The scale factor S6 was set to 0.75 because the GGA-PBE function was employed. An 11 × 11 × 1 k-point mesh was used for the interaction of the Brillouin-zone. The cutoff energy for the plane wave basis set was restricted to 400 eV, and a vacuum region of at least 12 Å was used in building the slab models. The convergence threshold was set as 10−4 eV in energy and 0.02 eV/Å in force, respectively. As shown in Fig. 0.5W0.5S2 on a monolayer 1T-SnS2 was constructed with a 2 × 2 supercell, which contains 24 Sn, 16 W and 80 S atoms, respectively. Three NH4+ ions were introduced in the four-layer 1T-Sn0.5W0.5S2 on a monolayer 1T-SnS2 system  were summarized in Supplementary Table a = 3.46 Å) was used in DFT calculations for the in-plane periodicity of the four-layer 1T-Sn0.5W0.5S2 on a monolayer 1T-SnS2 without and with intercalated NH4+ ions. To evaluate the stability of the adsorption of an acetone molecule on a three-layer 1T-SnS2 and three-layer 1T-Sn0.5W0.5S2 without and with intercalated NH4+ ions (each system contains a 4 × 4 supercell), the adsorption was defined by △Ea = Eacetone + Esubstrate − Eacetone–substrate  which is based on the density functional theory (DFT)Supplementary InformationPeer Review File


Under physiological conditions, vitamin C is the main antioxidant found in the central nervous system and is found in two states: reduced as ascorbic acid (AA) and oxidized as dehydroascorbic acid (DHA). However, under pathophysiological conditions, AA is oxidized to DHA. The oxidation of AA and subsequent production of DHA in neurons are associated with a decrease in GSH concentrations, alterations in glucose metabolism and neuronal death. To date, the endogenous molecules that act as intrinsic regulators of neuronal necroptosis under conditions of oxidative stress are unknown. Here, we show that treatment with AA regulates the expression of pro- and antiapoptotic genes. Vitamin C also regulates the expression of RIPK1/MLKL, whereas the oxidation of AA in neurons induces morphological alterations consistent with necroptosis and MLKL activation. The activation of necroptosis by AA oxidation in neurons results in bubble formation, loss of membrane integrity, and ultimately, cellular explosion. These data suggest that necroptosis is a target for cell death induced by vitamin C.  Under pFor many years, it has been thought that vitamin C could act as a pro-oxidant and kill tumor cells . In thisThe execution of necroptosis requires that caspase-8 (Casp8) be inhibited or absent . RIPK1 aNecroptosis was of particular interest in the current study because this type of death is induced in conditions of cerebral ischemia and reperfusion ,28,29, A−/- and SVCT2−/− neuronal cells and showed that ablation of MLKL delays cell death. Furthermore, if the uptake of AA is inhibited by the deletion of SVCT2, neuronal death is completely prevented. Our results suggest that the oxidation of vitamin C and the generation of DHA could regulate neuronal necroptosis under conditions of oxidative stress.In this study, we determined that the intracellular oxidation of AA favors the subsequent activation of necroptosis by phosphorylation of MLKL. Furthermore, we analyzed the disintegration characteristics in living cells by 4D microscopy, revealing that neuronal cells form extracellular vesicles prior to necroptotic disintegration in a manner similar to that recently reported in the literature . Finally22.1The detailed information on the compounds, antibodies and reagents is listed in 2.2l-glutamine (GIBCO) and 100 U/mL penicillin-streptomycin (GIBCO). HN33.11 cells were cultured using DMEM-HG (GIBCO) under the same conditions. To ensure mycoplasma-free cultures, the cells were treated with BM cyclin  prior to experiments. N2a(Mlkl−/−) and N2a(Svct2−/-) cells were generated by CRISPR/Cas9 using CAG-Cas9-2a-RFP and Cas9-ElecD plasmids  and transfection with Lipofectamine 3000 (Life Technologies). The gRNA target sequences for the murine initiation codons of MLKL and SVCT2 were GCACACGGTTTCCTAGACGC and TGTAGATCATATCCGACCTC, respectively. The cells were selected at 48 h posttransfection using a BD FACSAria III cell sorter. Single-cell RFP was sorted in 96-well plates. MLKL- and SVCT2-deleted colonies were verified by Western blotting. N2a-hSVCT2wt-EYFP, N2a-EGFP, HN33.11-hSVCT2wt-EYFP, and HN33.11-EGFP cells were generated by infection with lentiviral particles as previously described [Primary cortical neurons were obtained from rat embryos at 17 days gestation as previously described . All the2.32O2, the cover was removed, and the plates were placed in a live-cell perfusion chamber. Then, the cells were loaded with fluorescent probes for 10 min and washed with PBS. Finally, the cells were incubated in complete medium and imaged at 37 °C and 5% CO2 in a confocal spectral Zeiss LSM 780 live-cell system. The images were acquired in 4D  with an objective Plan-Apochromat 63x/1.40 Oil DIC M27. The following fluorescent probes were used: Hoechst 33342  350/461), Alexa Fluor 488 phalloidin  495/518), MitoTracker Red CMXRos  579/599), and cellmask (0.3X ex/em (nm) 650/655). Finally, the images were reconstructed in a movie using the Zen lite software (Zeiss).N2a and HN33.11 cells were seeded in 18-mm cover glasses in 12-well plates for 48 h. After treatment with H2.4Cells were seeded on coverslips. After treatment, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, washed with Tris-phosphate buffer  and incu2.52O2 for 30 min (or the concentration indicated in the figure). After this time, H2O2 was removed, and the cells were washed with PBS and incubated in complete medium for 3 h. Finally, cell viability was measured by XTT  colorimetric analysis. Cell death by loss of plasma membrane integrity was measured by flow cytometry (BD FACSAria III) with 500 nM TOPRO-3 (10 min) [2O2.N2a and HN33.11 cells were supplemented with 200 μM AA for 36 h. Then, intracellular oxidation of AA was induced by incubation with 500 μM H2.6The cells were trypsinized, resuspended in serum-free DMEM-F12 (GIBCO), incubated for 30 min with 500 nM CellROX Deep Red (Life Technologies) and analyzed by flow cytometry (BD FACSAria III). The flow cytometry data were processed with FlowJo software (Tree Star).2.7The cells were washed with PBS, trypsinized and resuspended in cold PBS. AA was measured using the ferric reducing (antioxidant) activity and ascorbic acid concentration (FRASC) colorimetric assay (bioassay system #EASC-100) according to the manufacturer's instructions.2.8N2a and HN33.11 cells were lysed with NP-40 buffer supplemented with a protease/phosphatase inhibitor cocktail . The proteins (30–50 μg) were separated using 10% SDS-PAGE gel, transferred to PVDF membranes  and probed with anti-SVCT2 (1:1000), anti-RIPK1 (1:3000) and anti-MLKL (1:5000) overnight. The membranes were then incubated with secondary antibodies, including HRP-conjugated donkey anti-goat IgG, donkey anti-rabbit IgG, donkey anti-rat IgG (1:6000) and anti-actin-HRP (1:50000) for 2 h at room temperature. The reaction was developed using the Western Lighting® Plus-ECL enhanced chemiluminescence substrate .2.9N2a and HN33.11 cells were trypsinized and fixed in 4% PFA at room temperature for 10 min. Then, the cells were permeabilized in 100% cold methanol for 30 min. The cells were washed twice by centrifugation in incubation buffer (0.5% BSA in Tris-phosphate buffer). For immunostaining and resuspended in 100 μL of incubation buffer with diluted RIPK1 (1:100) and MLKL (1:100) primary antibodies. After 1 h, the cells were washed by centrifugation and resuspended in Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (1:200) or Alexa Fluor 488 AffiniPure Donkey Anti-Rat IgG (1:200) secondary antibodies for 30 min. Finally, the cells were washed in incubation buffer, resuspended in PBS and analyzed by flow cytometry. The flow cytometry data were processed with FlowJo software (Tree Star).2.102 (Eppendorf). The primers used are listed in −ΔΔCt method.Total RNA from N2a and HN33.11 cells was purified using Trizol reagent . Real-time PCR was performed with Brilliant II SYBR® Green QPCR Master Mix . PCR  was carried out in Mastercycler Realplex2.11n of each analysis are described. In all cases, two-tailed Student's t-tests were used. p-values ≤0.05 were considered statistically significant. Data are presented as the means ± SEM.Statistical analyses were performed using GraphPad Prism version 6.01. In the legend of each figure, the exact values of 33.1in vitro cultures of the neuronal N2a and HN33.11 cell lines and cortical neurons with 200 μM AA for 36 h, as shown in 2O2 in N2a and HN33.11 cells and 50 μM H2O2 in cortical neurons for 30 min, after which the H2O2 was removed. The cells were left in complete medium for 3 h to perform the analyses  morphology is explained by the increase in the length of the z axis   A and B.F2O2 exhibited increased ROS levels over time (blue peak)  A and B. ue peak) A and B. ue peak) A and B, ver time C and D, ver time E and F, ver time  did not ver time G and H, 3.3AA is a cofactor of demethylases that regulates the expression of different genes . To exam2O2 treatment did not alter the distribution of necroptotic proteins. In N2a cells, H2O2 favored the expression of RIPK1 and MLKL  or necrostatin-1s (Nec-1s), an inhibitor of RIPK1 kinase activity. Interestingly, both Nec-1 and Nec-1s prevented neuronal death induced by AA oxidation in a dose-dependent manner . We repo3.5x, y, z and time). N2a, HN33.11 cells and cortical neurons maintained nuclear integrity, lost mitochondrial activity as measured by MitotrackerCMXRos stain and formed bubbles permeable to phalloidin prior to the loss of plasma membrane integrity  could affect the activity of enzymes important for the function of specific neurons in the CNS . Among t2O2 [p-ERK levels promote the expression of RIPK1, which may be associated with inhibition of apoptosis [−/− neurons were highly sensitive to oxidative stress, suggesting that SVCT2 could be a marker of resistance to cell death in the brain.It has long been assumed that "vitamin C" (without discriminating between AA and DHA redox states) can act as a pro-oxidant and induce cell death due to oxidative stress associated with a Fenton reaction and extracellular production of H2O2 ,10, ther2O2 ,26. Howe2O2 ,29. Intepoptosis , while ipoptosis . Interespoptosis ,52. Delepoptosis  and a glpoptosis . Thus, tpoptosis . Thus, Mpoptosis ,56. Our poptosis . Thus, Rpoptosis ,59. DHA poptosis ,56. In vpoptosis , it is cpoptosis ,34,60. ILF, MJB, PV, KS and FN conceived the experiments. LF performed the experiments; LF, MJB, PV, KS and FN analyzed the data; LF, PV and FN wrote the article; FN, KS and PV critically revised the manuscript. All authors approved the final version of the manuscript.None.


The radical-trapping antioxidant (RTA) activities of allicin and petivericin, thiosulfinates widely believed responsible for the medicinal properties of garlic and Petiveria, were determined in phosphatidylcholine lipid bilayers and mammalian cell culture. Petiveria, were determined in phosphatidylcholine lipid bilayers. The results indicate that both compounds are surprisingly ineffective, in sharp contrast with previous studies in organic solution which showed that they undergo facile Cope elimination to produce sulfenic acids – potent radical-trapping agents. In an effort to understand the medium dependence of this activity, a more lipophilic (hexylated) analog of petivericin was synthesized and shown to be among the most effective RTAs known, but only in the presence of a hydrophilic thiol (e.g. N-acetylcysteine). Additional symmetric and unsymmetric thiosulfinates were synthesized to shed light on the structural features that underlie this reactivity. These studies reveal that amphiphilic thiosulfinates which undergo S-thiolation with a hydrophilic thiol to give lipophilic sulfenic acids are required, and that an activated methylene group – key to promote Cope elimination – is not. Interestingly, the added thiol was also found to regenerate the sulfenic acid following its reaction with peroxyl radicals. This activity was diminished at more acidic pH, suggesting that it occurs by electron transfer from the thiolate. Allicin, petivericin and hexylated petivericin were assayed as inhibitors of lipid peroxidation in human TF1a erythroblasts and HEK-293 kidney cells, revealing similar efficacies in the low μM range – the same range in which allicin and petivericin were found to induce cell death concomitant with, or as a result of, glutathione (GSH) depletion. In contrast, hexylated petivericin was not cytotoxic throughout the concentration range assayed, and had no effect on GSH levels. Taken together, the results in lipid bilayers and in cell culture suggest that the greater lipophilicity of hexylated petivericin enables it to partition to membranous cell compartments where it forms a lipid-soluble sulfenic acid that traps peroxyl radicals, whereas allicin and petivericin partition to the cytosol where they deplete GSH and induce cell death.The radical-trapping antioxidant (RTA) activities of allicin and petivericin, thiosulfinates widely believed responsible for the medicinal properties of garlic and  Since its isolation by Cavallito and Bailey in 1944,The health benefits of garlic have been recognized since at least 2000 BC, making it the world's oldest known medicine.8 M–1 s–1.et al. reported that allicin reacts with lipid-derived peroxyl radicals with a rate constant of 1.6 × 105 M–1 s–1,6 M–1 s–1),31The medicinal properties of allicin are often ascribed to its controversial ‘antioxidant’ activity.e.g. an electron-rich phenolic moiety such as in α-TOH),via Cope elimination from allicin (eqn (1) and (2)).2), the analogous thiosulfinate derived from Petiveria alliacea – also known as the medicinal plant anamu found in South and Central America.1 and the phenylmethanesulfenic acid derived from 2 were too labile to study directly, investigations with the persistent 9-triptycenesulfenic acid 3 revealed that sulfenic acids have very weak O–H bonds ,k2 = 3 × 107 M–1 s–1).36Intrigued by the high reactivity of allicin toward peroxyl radicals despite the fact that it is devoid of any of the structural features common to good RTAs (4), which we found to be an excellent RTA in lipid bilayers, but only in the presence of a hydrophilic thiol – conditions that did not improve the poor reactivity of allicin and petivericin.S-thiolation of 4 by N-acetylcysteine (NAC) produces a lipophilic sulfenic acid which traps lipophilic peroxyl radicals and can be regenerated via reaction with another molecule of NAC at the bilayer interface. Herein we report the full details of these preliminary studies, and have in the interim expanded the scope of our investigations to: (1) elucidate the structural factors that contribute to the efficacy of hexylated petivericin as a RTA in lipid bilayers, (2) provide experimental support for our proposed mechanism, and (3) include a comparative study of the antioxidant activity and cytotoxicity of allicin, petivericin and hexylated petivericin in mammalian cell culture. Taken together, the results reveal that allicin and petivericin are not RTAs in cells, but instead deplete glutathione and induce cell death. In contrast, hexylated petivericin appears to be effective in cells; its differing behavior likely being due to its greater partitioning to the lipid bilayer.In a preliminary communication, we reported that allicin and petivericin are not particularly effective RTAs in lipid bilayers.I.1),2),3)4)n-alkylthiosulfinates 5 and 6 were prepared from the corresponding commercially available thiols via oxidation with I2 (5% solution in methanol), followed by oxidation with one equivalent of MCPBA, as shown in 7–10 were prepared by treatment of a thiol with sulfuryl chloride in acetic acid,Allicin (II.1), petivericin (2), 9-triptycenesulfenic acid (3) and the synthetic thiosulfinates (4–10) were determined in phosphatidylcholine liposomes using H2B-PMHC, a fluorogenic lipid oxidation probe.2B-PMHC is quenched by electron transfer from the PMHC moiety . Upon reaction with peroxyl radicals the PMHC moiety is no longer sufficiently electron-rich to quench the BODIPY (eqn (3)), leading to a rapid rise in its fluorescence.The RTA activities of allicin  was an excellent inhibitor  from a plot of ln[(I∞ – It)/(I∞ – I0)] vs. ln(1 – t/τ):Representative results are shown in either 1  or 2 , hereafter referred to as the ‘inhibited period’ (τ in eqn (4)), reflects the stoichiometry of the reaction of the added antioxidant and the peroxyl radicals. The inhibited periods in 3 yielding a slope of 12 ± 0.8 min μM–1. Since the dependence of the inhibited period on the concentration of α-TOH under similar conditions is roughly twice that of 3, and α-TOH is known to trap two peroxyl radicals,3 must trap only one MeOAMVN-derived peroxyl radical. A stoichiometry of one is consistent with the reactivity of compounds 1 and 2 in homogenous organic solution.4, a more lipophilic analog of petivericin (2), was able to retard the oxidation of H2B-PMHC  as a model thiol for these studies. While the addition of NAC did not impact the rate of oxidation of H2B-PMHC in the presence of 1 or 2  at 7.6 ± 0.2 min μM–1. Interestingly, when increasing concentrations of 4 are used with a constant concentration of NAC , significant activity was observed for some of these compounds in the presence of NAC. Representative results are shown in Since each of n-alkyl)thiosulfinates 5 and 6 were ineffective when co-administered with increasing amounts of NAC. However, the unsymmetrical thiosulfinates 7 and 8  were effective  the dependence of their inhibited periods on total antioxidant concentration , was essentially identical to 4 (7.6 ± 0.2 min μM–1). The two additional unsymmetrical thiosulfinates 9 and 10, which differ only in the sulfur atom to which oxygen is attached, differed markedly in their reactivity. Thiosulfinate 10, which is expected to react with NAC to yield the same sulfenic acid as that which arises in the S-thiolation of 4, displays similar activity, but with a lower relative apparent rate constant of 3.6 ± 0.8. In sharp contrast, thiosulfinate 9 is devoid of any radical-trapping activity.The lipophilic bis than the sulfenic acid alone . Representative data are presented for thiosulfinates III.sn-glycero-3-phospho-choline, PLPC). The hydroperoxide concentrations were determined using a phosphine–coumarin conjugate, which undergoes fluorescence enhancement upon oxidation to the phosphine oxide in the presence of hydroperoxides (eqn (5)).45Lipid hydroperoxide formation was also monitored directly in a select set of MeOAMVN-mediated oxidations of liposomes (at 37 °C) made exclusively from a polyunsaturated phospholipid  or by inhibition of glutathione peroxidase-4 (Gpx4) with RSL3.To probe the potential biological activity of hexylated petivericin, its efficacy in preventing lipid peroxidation was determined in human TF1a erythroblasts and HEK-293 embryonic kidney cells and compared to allicin and petivericin. The lipophilic C11-BODIPY50 values of 20 ± 1, 23 ± 2 and 74 ± 9 μM, respectively, when DEM was used as the initiator. Similar trends were observed in HEK-293 cells , hexylated petivericin seemed relatively more potent (EC50 = 39 ± 3 μM). Allicin and petivericin induced cell death at concentrations similar to those that were effective in inhibiting lipid peroxidation, i.e. TC50 = 39 ± 1 and 56 ± 3 μM, respectively, , petivericin (1.1 ± 0.1 μM) and hexylated petivericin (1.0 ± 0.1 μM).To demonstrate that each of allicin, petivericin and hexylated petivericin have similar inherent reactivity toward cellular thiols, we measured their potency as inhibitors of papain, the archetype cysteine protease. Papain was incubated with each thiosulfinate and its protease activity was then determined using a chromogenic substrate. The data (see ESIi.e. oxidation and condensation).4, an analog of petivericin which includes n-hexyl substitution at the 4-position of the phenyl ring. The requirement for Cope elimination of (4-hexylphenyl)methanesulfenic acid to account for the activity of 4 is evident in its diminished kinetics for radical-trapping (krel = 0.9 ± 0.1) relative to the persistent sulfenic acid 3 (krel = 25 ± 3); a difference which is fully consistent with that observed in chlorobenzene, where petivericin and 3 have kinh values of 2.0 × 105 and 30 × 105 M–1 s–1, respectively.Allicin and petivericin are effective RTAs in homogenous organic solution because they undergo Cope elimination to yield 2-propenesulfenic acid and phenylmethanesulfenic acid, respectively, and these molecules react with peroxyl radicals without an enthalpic barrier.S-thiolation reactions – with either a thiol or another molecule of thiosulfinate as the nucleophile.in vivo, with some tissues containing mM concentrations of glutathione, it is plausible that such an interaction may contribute to any potential RTA activity of allicin in biological contexts. Interestingly, while the addition of N-acetylcysteine had no impact on the RTA activity of either allicin or petivericin in liposomes, there was a significant increase in activity of hexylated petivericin. Since allicin  conversion of the thiosulfinate to the corresponding mixed disulfide 14. Nevertheless, the inhibited periods observed in 14 observed by LC/MS. Moreover, the addition of increasing amounts of NAC beyond one or two equivalents led to a concentration-dependent extension in the inhibited period, while maintaining the same overall kinetics – suggesting that NAC is able to regenerate the lipophilic sulfenic acid derived from 4 ,– and RSSR/RS– are 0.74 (vs. NHE in acetonitrile and water (pH 7),The regeneration of a lipid-soluble RTA using a water-soluble reducing agent is well-precedented. The most famous example is the combination of α-TOH and ascorbate;are 0.74  and –0.2are 0.74  V vs. NHS-thiolation (and not Cope elimination) is responsible for the formation of (4-hexylphenyl)methanesulfenic acid from 4 in lipid bilayers comes from studies of thiosulfinates that lack an activated methylene group adjacent the divalent sulfur atom – necessary for facile Cope elimination.n-octyl)thiosulfinate and bis(n-dodecyl)thiosulfinate were ineffective, the corresponding unsymmetrical thiosulfinates wherein either the octyl or dodecyl chain adjacent the divalent sulfur was replaced with an ethyl group displayed reactivities similar to the hexylated petivericin. Since the sulfenic acid that results from S-thiolation would be identical from thiosulfinates 5 and 7 (or 6 and 8), the lack of activity of 5 and 6 suggests that these compounds are too hydrophobic for efficient S-thiolation, which presumably takes place at the lipid/aqueous interface.Independent evidence that 9 and 10. Thiosulfinate 9, which is expected to undergo S-thiolation with NAC to produce ethanesulfenic acid was completely ineffective, while thiosulfinate 10, which is expected to undergo S-thiolation with NAC to yield (4-hexylphenyl)methanesulfenic acid, was similarly effective to hexylated petivericin. The disparate behaviour of 9 and 10 is illustrated in The importance of the lipophilicity of the sulfenic acid was demonstrated unequivocally by experiments with the unsymmetrical thiosulfinates 3 (12 ± 0.6) is more efficient than the regeneration of the sulfenic acids derived from the thiosulfinates 4 (7.6 ± 0.2), 7 (7.3 ± 0.4), 8 (6.6 ± 0.5) and 10 (8.4 ± 0.6). This difference reflects the relative persistence of the sulfinyl radicals formed following H-atom transfer from the sulfenic acids to peroxyl radicals. The sulfinyl radical derived from 3 is known to be persistent under the experimental conditions due to steric hindrance,2 and/or peroxyl radicals can compete with reduction by NAC. The pH dependence of the radical-trapping activities of the thiosulfinates further reinforces this point. While the apparent rates of radical-trapping do not vary significantly with pH, the dependence of the length of the inhibited period on antioxidant concentration decreases. This is consistent with slower regeneration of the sulfenic acid by the lower concentration of the thiolate form of NAC present at more acidic pH, allowing more time for deleterious side reactions.Comparison of the dependence of the inhibited periods as a function of added NAC suggests that the regeneration of the persistent sulfenic acid vide supra), no reports of cell-based assays of this activity can be found in the literature. Likewise for petivericin – although this is understandable since it was only relatively recently identified as the primary thiosulfinate in Petiveria.581/591 in TF1a erythroblasts . This suggests that the lipid peroxidation inhibition observed for allicin and petivericin may result simply from growth arrest and the resultant slowing  of aerobic metabolism associated with their toxicity. Accordingly, it would appear inappropriate to refer to allicin or petivericin as antioxidants in a biological context. In contrast, hexylated petivericin was not toxic throughout the concentration range examined in the lipid peroxidation assay (5–200 μM). This may be ascribed to its lipophilicity, which ensures localization to the lipid bilayer and thereby diminishes its reactivity with glutathione or nucleophilic cysteines on pro-apoptotic signaling proteins. The role of phase separation is supported by the lack of effect of hexylated petivericin on cellular thiol levels despite the results of proof-of-principle experiments wherein the three thiosulfinates were found to be equally potent inhibitors of papain, the archetype cysteine protease, when in homogenous solution.Since the concentrations of allicin necessary to prevent lipid peroxidation in our assays have been reported to induce death in some human cell lines, we determined the cytotoxicities of allicin, petivericin and hexylated petivericin toward the same human TF1a and HEK-293 cells used in the lipid peroxidation assays. These studies revealed that allicin and petivericin induced cell death at concentrations only marginally higher than those necessary to inhibit lipid peroxidation  by electron transfer from the corresponding thiolate. The results of experiments in human erythroblasts and embryonic kidney cells suggest that allicin and petivericin do not inhibit lipid peroxidation in cells, but induce cell death as a result of, or concomitant with, glutathione depletion. In contrast, hexylated petivericin is able to inhibit lipid peroxidation without inducing cell death or altering glutathione levels, presumably due to its more favourable partitioning to the lipid bilayer. The greater apparent activity of hexylated petivericin observed when lipid peroxidation is induced by Gpx4 inhibition with RSL3 suggests that the mechanism that operates in the lipid bilayers of liposomes extends to those that make up, and are found within, cells.The garlic-derived thiosulfinate allicin and the analogous secondary metabolite from the related species I.N-acetylcysteine, ascorbate, trypan blue, C11-BODIPY581/591 -4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid), RPMI-1640 media with/without phenol red, MEM media with/without phenol red, fetal bovine serum (FBS) and 7-aminoactinomycin D (7-AAD) were purchased from commercial sources and used as received. BCA protein assay kit was purchased from Thermo Scientific. Palmitoyl-2-linoleyl-sn-glycero-3-phosphocholine (PLPC) was synthesized according to the literature with purification by column chromatography immediately before use.2B-PMHC and the hydroperoxide probe in eqn (5) were prepared as described in 5–10 were prepared as described below.Egg phosphatidylcholine, penicillin–streptomycin, Dulbecco’s phosphate buffered saline (DPBS), phosphate buffered saline (PBS), MeOAMVN, m-Chloroperbenzoic acid (m-CPBA)  in dichloromethane (5 mL) was added dropwise at 0 °C. The mixture was stirred at 0 °C for one hour. Sodium carbonate (2 g) was added in small portions with vigorous stirring. The reaction mixture was stirred for an additional 1 h at 0 °C. The reaction mixture was then filtered through magnesium sulfate. The filtrate was concentrated under reduced pressure yielding crude product, which was purified by flash chromatography on silica gel (hexane : ethyl acetate = 5 : 1) to give compound 5 as a clear oil . 1H NMR : δ 3.06–3.20 , 1.73–1.87 , 1.27–1.51 , 0.85–0.89 ; 13C NMR : δ 56.20, 32.87, 31.72, 31.68, 30.82, 29.08, 28.97, 28.57, 23.42, 22.59, 22.57, 14.05; HRMS (EI+) calcd for C16H35S2O [M + H]+ 307.2129, obsd 307.2135.To a solution of octane-1-thiol  in MeOH (10 mL) at 0 °C, Iodine solution (5% in methanol) was added dropwise till the reddish color of iodine stayed, and then keeping the reaction stirring at 0 °C for another 20 min. Sodium thiosulfate was added to quench the excess iodine. The mixture was evaporated to remove the methanol mostly. The residue was treated with water (10 mL) and the resulting solution was extracted with ether. The extracts were combined, dried over magnesium sulphate and vacuumed to give a oil, which was dissolved in DCM (10 mL) at 0 °C. 5 and isolated as a white solid by recrystallization from ether . 1H NMR : δ 3.06–3.20 , 1.73–1.87 , 1.27–1.51 , 0.85–0.89 ; 13C NMR : δ 56.20, 32.87, 31.72, 31.68, 30.82, 29.08, 28.97, 28.57, 23.42, 22.59, 22.57, 14.05; HRMS (EI+) calcd for C12H25S [M – C12H25SO]+ 201.1677, obsd 201.1674; calcd for C12H25SO [M – C12H25S]+ 217.1626, obsd 217.1652.The title compound was obtained as for 7 as a light yellow oil . 1H NMR : δ 3.26–3.21 , 3.09 , 1.89–1.79 , 1.39 , 1.33–1.21 , 0.82 ; 13C NMR : δ 62.8, 31.6, 30.6, 28.9, 28.8, 27.9, 23.4, 22.5, 15.1, 14.0; HRMS (EI+) calcd for C8H17SO [M – C2H6S]+ 161.1000, obsd 161.1071.A well-stirred mixture of octane-1-thiol  and acetic acid  is cooled to –20 °C. Sulfuryl chloride  is added dropwise over a period of 20 min. Gas evolution is observed during the addition. Stirring is continued for 30 min at –20 °C, and then the mixture was allowed to warm up to room temperature over a period of 2 h and stirred at room temperature for 2 h. Acetyl chloride is vacuumed off to leave the sulfinyl chloride. To a solution of sulfinyl chloride  in DCM (10 mL), pyridine  was added dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, to which ethanethiol  in DCM (1 mL) was added dropwise. After stirring at 0 °C at 1 h, water (10 mL) was added slowly to quench the reaction. The organic layer was separated and water phase was extracted with ether (15 mL × 6). All the organic phase was combined and washed by HCl  and brine (20 mL). The organic solvent was then removed under reduced pressure. The resulting oil was purified by flash chromatography on silica gel (hexane : ethyl acetate = 3 : 2) to give compound 7 following flash column chromatography on silica gel (hexane : ethyl acetate = 3 : 1) as a light yellow oil . 1H NMR : δ 3.26–3.21 , 3.09 , 1.89–1.79 , 1.39 , 1.33–1.21 , 0.82 ; 13C NMR : δ 62.7, 31.8, 30.6, 29.5, 29.4, 29.2, 29.1, 28.9, 27.9, 23.4, 22.6, 15.1, 14.0; HRMS (EI+) calcd for C12H25SO [M – C2H6S]+ 217.1626, obsd 217.1530.The title compound was obtained as for 7 following recrystallization from diethylether as a white solid . 1H NMR : δ 7.20–7.38 , 4.48 , 2.76 , 2.62 , 1.65–1.55 , 1.35–1.26 , 1.20 , 0.88 ; 13C NMR : δ 144.4, 131.2, 128.9, 124.9, 68.7, 35.6, 31.6, 31.2, 31.0, 28.8, 22.5, 14.8, 14.0; HRMS (EI+) calcd for C2H5S2O [M – C13H19]+ 108.9782, obsd 108.9764, calcd for C13H19 [M – C2H5S2O]+ 175.1487, obsd 175.1507.The title compound was obtained as for 7 following flash column chromatography on silica gel (hexane : ethyl acetate = 3 : 1) as a white solid . 1H NMR : δ 7.14–7.29 , 4.31 , 2.93 , 2.59 , 1.65–1.54 , 1.33–1.26 , 0.88 ; 13C NMR : δ 143.2, 132.2, 129.0, 128.9, 57.2, 40.3, 35.6, 31.6, 31.3, 28.8, 22.6, 14.0, 8.1; HRMS (EI+) calcd for C13H18S [M – C2H5SOH]+ 206.1129, obsd 206.1107.The title compound was obtained as for II.Egg phosphatidylcholine (Egg PC) or PLPC (75 mg) was weighed in a dry vial and dissolved in a minimum volume of chloroform. The solvent was then evaporated under argon to yield a thin film on the vial wall. The film was left under vacuum for 2 hours to remove any remaining solvent. The lipid film was then hydrated with 4.0 mL of a 10 mM phosphate-buffered saline (PBS) solution (pH 7.4 or 5.8) containing 150 mM NaCl, yielding a 24 mM lipid suspension. The lipid suspension was then subjected to 10 freeze-thaw-sonication cycles, followed by extrusion using a mini-extruder equipped with a 100 nm polycarbonate membrane.III.2B-PMHC in acetonitrile (13 μM). Each resultant solution was then diluted to 1 mL with PBS, from which 280 μL of each was loaded into a well of a 96-well microplate. The solution was equilibrated to 37 °C for 5 min, after which 20 μL of a solution of 3 mM in 2,2′-azobis- (MeOAMVN) in acetonitrile was added to each well using the reagent dispenser of a microplate reader. The fluorescence was then monitored for 6 h at 50 s time intervals . The final solutions in each well were 1 mM in lipids, 0.15 μM in H2B-PMHC, 0.2 mM in MeOAMVN and either 4.5, 9, 13.5, 18, 22.5 μM in antioxidant. Each liposome contained, on average, 15 fluorophores with an Egg PC/fluorophore molar ratio of 6700 : 1. Under these conditions, no fluorescence self-quenching occurs within the liposome bilayer.To individual 45 μL aliquots of the 24 mM liposome solution were added increasing amounts  of a solution of the test antioxidant in acetonitrile (0.15 mM) and 10 μL of a solution of HIV.11 in acetonitrile (160 mM) was added to each well and the initial rate of the reaction was obtained by measuring the fluorescence  for 50 s using a microplate reader. The lipid hydroperoxide concentration was calculated based on the initial rate of the reaction.Stock solutions of the different antioxidants in acetonitrile were added into various amounts of unilamellar PLPC liposomes in pH 7.4 buffer. MeOAMVN (48 μL of a 2.3 mM solution in acetonitrile) was then added to initiate lipid peroxidation. The reaction mixtures were stirred at 37 °C in an aluminium heating block. The final concentrations of PLPC and MeOAMVN were 8.75 mM and 0.15 mM, respectively. Every 12 minutes, 10 μL of the reaction mixture was withdrawn and transferred to a well of a 96-well microplate and 165 μL of MeOH containing butylated hydroxytoluene 45 mM) was added to destroy the liposome and prevent adventitious oxidation. Using the reagent dispenser of the microplate reader, 25 μL of a solution of  mM was aV.5 cells mL–1 or 1 × 106 cells mL–1) were treated with each of the thiosulfinates 1, 2 and 4 at final concentrations from 5 μM to 200 μM and incubated at 37 °C for 22 hours in phenol red-free RPMI-1640 media with 10% FBS and 1% penicillin–streptomycin for TF1a cells and phenol red-free MEM media with 10% FBS, 1% non-essential amino acid, 1 mM sodium pyruvate and 1% penicillin–streptomycin for HEK-293 cells in a humidified atmosphere containing 5% CO2 in air. Cells were then treated with 1 μM C11-BODIPY581/591 in media and incubated at 37 °C in the dark for 30 minutes after which lipid peroxidation was initiated with either diethylmaleate (9 mM) for 2 hours or -RSL3 (4 μM) for 6 hours. Treated TF1a cells were then collected by centrifugation at 300×g for 3–4 minutes, whereas treated HEK-293 cells were then washed by DPBS, detached with accutase followed by centrifugation at 300×g for 3–4 minutes. Cells were resuspended in DPBS and analyzed by flow cytometry at a final concentration of 1 × 106 cells mL–1 . Cells that were not treated with DEM/RSL3 were used as negative control. Cells that were not treated with thiosulfinates were used as positive control.TF1a cells were cultured in RPMI-1640 media with 10% FBS and 1% penicillin–streptomycin. HEK-293 cells were cultured in MEM media with 10% FBS, 1% non-essential amino acid, 1 mM sodium pyruvate and 1% penicillin–streptomycin. Cells (5 × 10VI.5 cells mL–1) were treated with each of thiosulfinates 1, 2 and 4 at final concentrations ranging from 5 μM to 200 μM and incubated at 37 °C for 22 hours in phenol red-free RPMI-1640 media with 10% FBS and 1% penicillin–streptomycin for TF1a cells and phenol red-free MEM media with 10% FBS, 1% non-essential amino acid, 1 mM sodium pyruvate and 1% penicillin–streptomycin for HEK 293 cells in a humidified atmosphere containing 5% CO2 in air. Treated HEK 293 cells were washed with DPBS followed by accutase to detach from the plate. Cells were collected into the flow cytometry tubes and then treated with 7-AAD (5 μL of a 50 μg mL–1 solution/1 × 106 cells) for 10 minutes and analyzed by flow cytometry at a final concentration of 5 × 105 cells mL–1 . Cell viability was also determined using hemocytometry with trypan blue. After incubation of TF1a cells (5 × 105 cells mL–1) with each of 1, 2 and 4 for 22 hours, 50 μL of 0.4% trypan blue solution was added into 350 μL of cells. The cells were counted using a hemocytometer under light microscopy. The cells which excluded the probe were considered viable. About 200–250 cells were counted for each sample.TF1a cells were cultured in RPMI-1640 media with 10% FBS and 1% penicillin–streptomycin. HEK 293 cells were cultured in MEM media with 10% FBS, 1% non-essential amino acid, 1 mM sodium pyruvate and 1% penicillin–streptomycin. Cells (5 × 10VII.5 cells mL–1) and HEK-293 (5 × 105 cells mL–1) cells were treated with each of the thiosulfinates 1, 2 and 4 at final concentrations from 10 μM to 200 μM separately and incubated at 37 °C for 22 hours in phenol red-free media in a humidified atmosphere containing 5% CO2 in air. Cells were then lysed with HEPES hypotonic buffer and the cytosolic fraction was collected. Intracellular thiol concentration was determined by absorbance at 412 nm using the glutathione colorimetric assay by titration with Ellman's reagent  using glutathione as a standard. Intracellular protein concentration was determined using a commercial Bradford assay kit.TF1a (5 × 10VIII.Nα-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BAPNA) was first dissolved in a minimal amount of DMSO and diluted in buffer to a final concentration of 1 mM. The wells of a 96 well microplate were loaded with buffer, papain solution  and thiosulfinate solution to a final volume of 160 μL. The microplate was then incubated at 37 °C for 30 minutes. BAPNA  was added by the reagent dispenser of the microplate reader and the activity of papain was measured at 37 °C by monitoring the production of p-nitroaniline by absorbance at 410 nm every 5 seconds for 5 minutes.Papain (25 μM) was incubated with DTT (1 mM) for 30 minutes at 0 °C in pH 6.1 buffer . The small molecules were then removed by filtration through an 10 kDa Amicon Filter. Stock solutions of the various thiosulfinates in acetonitrile were prepared directly before use. The chromogenic substrate Supplementary informationClick here for additional data file.


However, the physiological function of SLC41A1 remains to be elucidated. In this study, cellular Mg2+ transport assays combined with zebrafish slc41a1 knockdown experiments were performed to disclose SLC41A1 function and its physiological relevance. The gene slc41a1 is ubiquitously expressed in zebrafish tissues and is regulated by water and dietary Mg2+ availability. Knockdown of slc41a1 in zebrafish larvae grown in a Mg2+-free medium resulted in a unique phenotype characterized by a decrease in zebrafish Mg content. This decrease shows that SLC41A1 is required to maintain Mg2+ balance and its dysfunction results in renal Mg2+ wasting in zebrafish larvae. Importantly, the Mg content of the larvae is rescued when mouse SLC41A1 is expressed in slc41a1-knockdown zebrafish. Conversely, expression of mammalian SLC41A1-p.Asp262Ala, harboring a mutation in the ion-conducting SLC41A1 pore, did not reverse the renal Mg2+ wasting. 25Mg2+ transport assays in human embryonic kidney 293 (HEK293) cells overexpressing SLC41A1 demonstrated that SLC41A1 mediates cellular Mg2+ extrusion independently of sodium (Na+). In contrast, SLC41A1-p.Asp262Ala expressing HEK293 cells displayed similar Mg2+ extrusion activities than control (mock) cells. In polarized Madin-Darby canine kidney cells, SLC41A1 localized to the basolateral cell membrane. Our results demonstrate that SLC41A1 facilitates renal Mg2+ reabsorption in the zebrafish model. Furthermore, our data suggest that SLC41A1 mediates both Mg2+ uptake and extrusion.Solute carrier family 41 member A1 (SLC41A1) has been suggested to mediate magnesium (MgThe online version of this article (10.1007/s00424-018-2234-9) contains supplementary material, which is available to authorized users. Of the 2400 mg of Mg2+ filtered in the kidney on a daily basis in humans, 95–99% is recovered through tubular Mg2+ reabsorption [2+ handling results in a heterogeneous group of disorders that are characterized by hypomagnesemia (serum Mg2+ < 0.7 mmol/L) [2+ wasting has resulted in the elucidation of the molecular mechanisms that regulate renal Mg2+ reabsorption [Magnesium  and thick ascending limb of Henle’s loop . The remainder is reabsorbed in the distal convoluted tubule (DCT), where Mg2+ reabsorption takes place in a transcellular manner via transient receptor potential melastatin type 6 (TRPM6) Mg2+ channels [TRPM6 cause hypomagnesemia and secondary hypocalcemia (HSH) [2+ reabsorption. However, the basolateral Mg2+ extrusion mechanism remains subject of debate, and no proteins have been characterized facilitating this process [Mgchannels . Mutatiobule PT, % and thiia (HSH) . Its memule PT, 2% and thiia (HSH) , 23, 26. process .2+ extrusion by operating as a Na+-Mg2+ exchanger [2+ transporter and is widely expressed in the human body [2+ was shown to regulate renal Slc41a1 gene expression in mice [2+ transport, SLC41A1 has not been linked with the regulation of the Mg2+ balance in vivo, remaining unclear whether SLC41A1 is relevant for renal Mg2+ reabsorption.Recently, the solute carrier family 41 member A1 (SLC41A1) was suggested to facilitate cellular Mgxchanger , 20. SLCman body , 28. In man body , and tarman body . In addi in mice . Despite2+ transport experiments.In this study, we aimed to elucidate the physiological role of SLC41A1 by knockdown experiments in the zebrafish model. Moreover, the molecular function of SLC41A1 was studied by cellular MgSlc41a1 transcript was amplified from a cDNA clone with GenBank accession number NM_173865  using Phusion polymerase. The primer sequences contained AscI and XbaI and are provided in Table AscI and XbaI restriction sites. The p.Asp262Ala mutation in mouse SLC41A1  was introduced using the QuikChange site-directed mutagenesis kit . For experiments in the zebrafish model, the SLC41A1 constructs were subcloned into pT7Ts vectors using the AscI and XhoI restriction enzymes. All constructs were verified by sequence analysis. Throughout the manuscript, we followed the guidelines from HUGO, MGI, and ZFIN on gene nomenclature for human, mouse, and zebrafish genes, respectively.The full-length mouse slc41a1 in adult zebrafish tissues, three females and three males were dissected following anesthesia (0.1% (v/v) 2-phenoxyethanol ). Brain, ovary, gills, testis, heart, spleen, kidney, gut, operculum, scales, and liver tissues were collected and stored at − 80 °C until analysis. The expression of slc41a1 in the pronephric tissue was determined in 120 hours post-fertilization (hpf) zebrafish which were grown in E3 medium . After anesthesia with tricaine/Tris pH 7.0 solution, the pronephros was isolated from zebrafish larvae as previously reported [Zebrafish from the Tupfel long-fin (TLF) strain were used for experimentation. For the study of the tissue distribution of reported . Samplesslc41a1 during zebrafish development, zebrafish were grown in E3 medium and sampled at 6, 12, 24, 48, 72, 96, and 120 hpf. For sampling, animals were anesthetized with tricaine/Tris pH 7.0 solution. Each sample was constituted of 10 embryos/larvae and was stored at − 80 °C until further analysis.For the study of the expression of slc41a1 gene expression by dietary and water Mg2+ in zebrafish gills and kidney, fish Mg2+ balance was challenged by feeding different Mg2+ diets  Mg) or by exposure to different water Mg2+ concentrations  as previously reported [v/v) 2-phenoxyethanol . After anesthesia, death of animals was induced by spinal transaction and organs were collected, immediately frozen in liquid nitrogen, and stored at − 80 °C until analysis.To study the regulation of slc41a1 gene expression and of the zebrafish orthologs of human magnesiotropic genes expressed in the DCT  in 120 hpf zebrafish, zebrafish embryos were grown in different Mg2+-containing E3 mediums from fertilization until 120 hpf, time at which sampling took place. The different Mg2+-containing E3 mediums had the following electrolyte concentrations (in mmol/L): low Mg2+ water, 5 NaCl, 0.17 KCl, 0.33 CaCl2; control Mg2+ water, 0.33 MgSO4, 5 NaCl, 0.17 KCl, 0.33 CaCl2; high Mg2+ water, 25 MgSO4, 5 NaCl, 0.17 KCl, 0.33 CaCl2. At 120 hpf, zebrafish were anesthetized with tricaine/Tris pH 7.0 solution and samples (10 larvae/sample) were collected and stored at − 80 °C until further analyses.To study the regulation of slc41a1 transcripts were used: a translation blocking MO 5′-TGATCTTTCAACCAGAGTACCATGC-3′ and a splice-site blocking MO 5′-GAAGGACTCAATGGTCTCACCTAAA-3′ . In parallel with these slc41a1-MOs, a standard mismatch MO directed (control-MO) against a human β-globin intron mutation, 5′-CCTCTTACCTCAGTTACAATTTATA , was used in control zebrafish. MOs were diluted in deionized, sterile water supplemented with 0.5% (w/v) phenol red and injected in a volume of 1 nL into the yolk of one- to two-cell stage embryos using a pneumatic PicoPump pv280 . WT embryos (uninjected) were also included in the experiments to control for the effects of the injection procedure per se. After injection, embryos from the same experimental condition were placed in three Petri dishes and cultured at 28.5 °C in Mg2+-free E3 medium  or normal E3 medium containing 0.33 mmol/L MgSO4, which was refreshed daily. Three different types of experiments were carried out: a dose-response experiment , a time-course experiment , and an experiment to study the gene expression of slc41a1, trpm6, slc12a3, slc41a3, cnnm2a, cnnm2b, pro-egf, and fxyd2 . During samplings, animals were anesthetized with tricaine/Tris pH 7.0 solution, and morphologic phenotypes were analyzed under the microscope . Two morphologic phenotypes were distinguished: normal and malformed . After phenotyping, only morphologically normal fish were sampled, each sample constituted of 10 larvae, which were stored at − 80 °C until further analyses.Zebrafish eggs were obtained from natural spawning of wild-type (WT) TLF zebrafish which were bred and raised under standard conditions (28.5 °C and 14 h of light:10 h of dark cycle) in accordance with international and institutional guidelines. Two non-overlapping morpholinos (MOs) targeting slc41a1-MO as described above. At 120 hpf, zebrafish were anesthetized with tricaine/Tris pH 7.0 solution, and morphologically normal zebrafish were sampled. Samples, constituted by 10 larvae/sample, were stored at − 80 °C until further analyses.In vivo cRNA rescue experiments were performed with mouse WT SLC41A1 and mutant SLC41A1-p.Asp262Ala cRNAs. Constructs were subcloned into the pT7Ts expression vector, suitable for rescue experiments in zebrafish , and cRN2+ and Ca2+. The washing procedure was repeated twice. Fish were then dried and digested as described previously [Sample processing started by quickly washing the samples with nanopure water in order to avoid contamination of remaining water Mgeviously . Within-RNA was isolated from zebrafish adult tissues, the pronephros, and total embryos/larvae using TRIzol reagent  according to the manufacturer’s instructions, in which glycogen  was used in order to maximize the RNA recovery. One microgram of RNA (200 ng in the case of the pronephric samples) was subjected to DNase treatment to prevent genomic DNA contamination and subsequently used to perform the reverse transcriptase reaction.elongation factor-1α (elf1α) [−ΔΔCt). Primer sequences are shown in Table Changes in target genes mRNA levels were determined by relative real-time quantitative polymerase chain reaction (RT-qPCR) as previously described  and in a (elf1α) . Relativslc41a1-MO, cDNA from 120 hpf control (injected with 16 ng control-MO) and morphant (injected with 16 ng splice-site blocking slc41a1-MO) larvae was used for PCR analysis. Three different primer pairs flanking exon 5 were used (Table w/v) agarose gel. The efficacy of the splice-site blocking MO targeting exon 5 (145 bp) in the slc41a1 gene was measured by RT-qPCR following the methods described above. The specific primers used are shown in Table slc41a1-MO. Thus, the resulting amplicon quantification reflects the expression levels of the functional, non-spliced slc41a1. In addition, slc41a1 expression of the total slc41a1 transcript, which entails non-spliced and aberrantly spliced variants, was measured by RT-qPCR (primers used are shown in Table To determine the knockdown mechanism induced by the splice-site blocking v/v) fetal calf serum , 2 mmol/L L-glutamine, and 10 μg/mL non-essential amino acids, at 37 °C in a humidity-controlled incubator with 5% (v/v) CO2. The cells were transiently transfected with the respective DNA constructs using Lipofectamine 2000  at 1:2 DNA:Lipofectamine ratio for 48 h unless otherwise stated.HEK293 cells were grown in Dulbecco’s modified Eagle’s medium  containing 10% (v/v) fetal calf serum , 2 mmol/L L-glutamine, ciproxin, and 10 μg/mL non-essential amino acids, at 37 °C in a humidity-controlled incubator with 5% (v/v) CO2. The cells were stably transfected with SLC41A1 using Metafectene pro (Biontex) in 1:3 DNA:Metafectene ratio. After 48 h, transfected cells were placed on selection medium containing 0.8 mg/mL G418 and 0.15 mg/mL hygromycin for 14 days.Madin-Darby kidney (MDCKI) cells were cultured in DMEM with 5% (6 cells/12 wells) and transfected with empty (mock), WT, and mutant SLC41A1 constructs for 48 h and seeded on poly-L-lysine (Sigma)-coated 12-well plates. For extrusion experiments, the cells were transferred to Mg2+-free medium supplemented with 1 mmol/L 25Mg2+ during the last 24 h . Before the start of the experiment, cells were washed three times in basic uptake buffer  and subsequently placed in basic uptake buffer with 0.5 mmol/L Mg2+  for 5, 10, or 15 min. For experiments using NMDG, NaCl was replaced in the buffer by NMDG (pH 7.4 with HCl), and in the Cl-free experiments, NaCl, KCl, and CaCl2 were replaced by Na-gluconate, K-gluconate, and Ca-gluconate (pH 7.4 with NaOH). The buffer was removed and subjected to inductively coupled plasma mass spectrometry (ICP-MS) analysis. Uptake experiments were performed as described previously [25Mg content of the cells was determined at time point 0 and was subtracted from the other time points when calculating net fluxes. In the experiments using pharmacological inhibition, 100 μM quinidine, 100 μM ouabain, and 100 μM 2-APB were added 30 min before the start of the uptake. The coefficient of variation (CV) for intra-assay and inter-assay variability has been calculated to determine the variability of our assay. The intra-assay CV is 4% and the inter-assay CV is 12%.HEK293 cells were seeded in 12-well plates  primary antibody and peroxidase-conjugated sheep anti-mouse secondary antibodies .w/v) paraformaldehyde solution in PBS, permeabilized in 0.3% (v/v) Triton in PBS for 15 min, and quenched for 15 min in 50 mM NH4Cl. After blocking with goat serum (16% (v/v) for 30 min), cells were incubated with primary antibody rat anti-HA  or anti-E-Cadherin  for 60 min. After rinses in PBS, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG or Alexa Fluor 594-conjugated donkey anti-rabbit IgG secondary antibodies  and DAPI for 45 min. After PBS and ethanol rinses, the cells were mounted on slides using Mowiol. Fluorescence microscopy was performed with a Zeiss inverted microscope, and images were taken with ImageJ software.SLC41A1-expressing MDCKI cells were seeded on 0.4 μm transwell filters (Corning Costar) and cultured for 7 days. Subsequently, the cells were rinsed with PBS and fixed for 30 min with 4% (slc41a1 expression in the pronephros), an unpaired Student’s t test was used. Statistical significance was accepted at P ˂ 0.05.All results are depicted as mean ± standard error of the mean (S.E.M.). Statistical analyses were conducted by one-way ANOVA. Tukey’s post-test was used to identify significantly different groups. When only two experimental groups were present X4PX6D, which form the Mg2+ selective pore [To examine the involvement of SLC41A1 in Mg nephron , 19, 30.ive pore , are preslc41a1 gene is ubiquitously expressed in adult tissues , similarly to the orthologs of mammalian DCT-specific genes such as trpm6, slc41a3, and cnnm2a  slc41a1 transcripts , the morphology of slc41a1 morphants was also comparable to controls  [2+ extrusion in cells expressing SLC41A1-p.Asp262Ala was comparable to mock  . Indeed,ock Fig. b. Cells ing Fig. c. Plasmaing Fig. d. To exaons Fig. e. To exc2+ extrusion was not Na+-dependent in our setup, we examined whether SLC41A1 can facilitate Mg2+ uptake despite high extracellular Na+ concentrations using physiological conditions of 1 mM Mg2+ and 140 mM Na+ in the extracellular buffer. Indeed, SLC41A1 increases 25Mg2+ uptake fivefold compared to mock-transfected cells  Mg2+ wasting since the pronephros is the only exiting route for Mg2+ in developing zebrafish (0–120 hpf). In this time frame, there is no intestinal Mg2+ excretion since zebrafish do not eat and also do not drink as they are hyperosmotic with respect to the surrounding environment. In addition, by growing the zebrafish in a Mg2+-free E3 medium, skin Mg2+ uptake is abolished, so the lower Mg content in slc41a1 morphants compared to controls can only be explained by deficient tubular Mg2+ reabsorption. In agreement with a defect in renal Mg2+ reabsorption, the total Mg content in slc41a1 morphants was decreased after full maturation of the pronephros (96 and 120 hpf) but not at earlier time points [The low Mg content in zebrafish e points .2+ extrusion function. As SLC41A1 is expressed in the DCT [2+ occurs, it can be reasoned that SLC41A1 may contribute to the extrusion of Mg2+ in this segment of the nephron. In line with these findings, mRNA expression of genes involved in DCT Mg2+ reabsorption was upregulated in slc41a1 morphants , fu2+ leak in slc41a1 morphants could not be explained by glomerular dysfunction and/or disturbances in renal fluid flow, because enlarged pericardial cavities (fluid retention) and pronephric cysts were absent in the fish where the total Mg content was measured. These kidney defects have been, however, associated with slc41a1 knockdown in a study by Hurd et al. [slc41a1 morphants.The renal Mgd et al. . Differe2+ wasting phenotype in slc41a1-knockdown zebrafish was rescued by expression of mouse WT SLC41A1 but not by expression of SLC41A1-p.Asp262Ala, harboring a mutation in a key residue for SLC41A1 function [2+ transport function of SLC41A1 is required to rescue the phenotype and that zebrafish Slc41a1 is functional equivalent to mammalian SLC41A1.The renal Mgfunction . This im2+ transporter that facilitates cellular Mg2+ uptake as well as Mg2+ extrusion dependent on the concentration gradient. In contrast to previous studies suggesting that SLC41A1 functions as a Na+-Mg2+ exchanger, our results do not support a Na+-dependent mechanism [2+ in Na+-free (NMDG-containing) buffer which cannot be blocked by Na+-channel blocker quinidine and Na+-K+-ATPase inhibitor ouabain.In line with SLC41A1 function in vivo, we demonstrate that SLC41A1 is a Mgechanism , 20. Our2+ transport remains elusive. SLC41A1 function is not altered by ouabain, in line with an electroneutral transport mode and a Na+-independent mechanism. Our experiments exclude Cl− as co-transported ion, but we were not able to identify the counter/co-ion. Based on the strong capacity of SLC41A1 to mediate both Mg2+ uptake and extrusion, the Mg2+ gradient seems to be the main determinant of Mg2+ transport. Previous studies have suggested the presence of a Mg2+-Mg2+ exchange mechanism in erythrocytes, where Mg2+ can compete with Na+ extracellularly for common binding sites in the erythrocyte Na+-Mg2+ exchanger [2+ exchange and its physiological relevance is questionable, we cannot exclude this possibility based on our data. It is interesting to note that the fivefold increase in Mg2+ uptake by SLC41A1 is high compared to previous experiments measuring CNNM2-dependent Mg2+ uptake (2–3-fold). In contrast to CNNM2-dependent Mg2+ uptake, the effects cannot be blocked by 2-APB, supporting that SLC41A1 mediates Mg2+ uptake and is not dependent on TRPM7.The molecular mechanism of SLC41A1-mediated Mgxchanger , 14. AltSLC41A1 mutation has been previously associated with a nephronophthisis-like phenotype [SLC41A1 mutation that developed renal failure, eventually requiring renal transplantation [2+ transport impairment. However, it must be indicated that kidney failure typically results in Mg2+ conservation [2+ reabsorption defects in these specific SLC41A1 patients. In contrast to the patient’s phenotype, no signs of renal cysts or dilatations were observed in our zebrafish model though our experiments were restricted to developing zebrafish (0–120 hpf). Furthermore, Mg2+ supplementation in the medium where zebrafish morphants were grown did not restore the Mg content to comparable levels as in controls. Similarly, in patients with inherited Mg2+ reabsorption disturbances and without renal cysts, Mg2+ homeostasis is not restored upon Mg2+ supplementation [An exon-skipping henotype . This stantation . These pervation , an effeslc41a1 knockdown with both splice-site blocking and translation blocking MOs, a feature that was rescued by expression of mammalian SLC41A1. These results clearly demonstrate the specificity of the slc41a1 knockdown in zebrafish to yield Mg2+ deficiency and discard these results as off-target effects of the slc41a1 MO methodology used in the present study. A limitation is the use of HEK293 cells for our functional assays. However, Mg2+-transporting DCT cell lines are not available and our approach is more physiological than previous experiments. All measurements were performed in physiological Mg2+ concentrations (0.5–1 mM). Previous experiments were dependent on fluorescent probes and required super-physiological Mg2+ concentrations to load the cells (10 mM) [2+ stores. An additional limitation of our study is that we cannot exclude differences in buffer capacity of the cells induced by overexpression of SLC41A1.A strength of our study is that the reduced Mg content was present when inducing the  (10 mM) , 20. The2+ balance by transporting Mg2+ in the kidney. Further characterization of SLC41A1 in renal cells is required to determine the molecular mechanism of SLC41A1-mediated Mg2+ transport. Based on the SLC41A1 function disclosed, patients with hereditary hypomagnesemia should be screened for SLC41A1 mutations.In conclusion, our data indicate that SLC41A1 regulates the body MgESM 1(PDF 629 kb)


Dear Editor,RCAN1) gene is highly expressed in human hearts and brains  is a major birth defect worldwide. However, the aetiology of CHD remains unclear and the detailed molecular mechanisms underlying CHD pathogenesis have not been fully understood. The human regulator of calcineurin RCAN gene is To investigate if the RCAN1.4 plays a role in heart development, we evaluated the expression of the RCAN1.1 and RCAN1.4 proteins in E10.5 mouse embryonic hearts and human hearts. The results revealed that both the RCAN1.1 and RCAN1.4 were expressed in mouse embryonic hearts and human hearts . Eight SNPs with a minor allele frequency (MAF) > 0.05 were identified within the −2 kb of 5′ regulatory region of RCAN1.1 and RCAN1.4 transcripts. Three SNPs  and the other five  were positioned in 5′ regulatory regions of RCAN1.1 and RCAN1.4 transcripts, respectively  in the coding region of RCAN1.4. Among them, only the genotype distribution of SNP rs2243890 A>G (HGVS Name: NM_203418:c.-1712 A>G) located in RCAN1.4 promoter was normally different between the CHDs and control (Table S2). We observed the higher frequencies of G allele in CHD (MAF = 0.09) than control (MAF = 0.05)  (Table S2). To further confirm the validity of the association of rs2243890 A>G with CHD, we not only verified the status of rs2243890 A>G by re-sequencing a 0.5-kb genomic sequence around the SNP in the previously analysed 412 sporadic CHD patients and 213 matched controls, but also examined the same sequence in 458 newly collected sporadic CHD patients and 1,107 healthy controls from Shanghai by Sanger sequencing. Consistent results were obtained for this SNP  (Table P = 4.68E−05) (Table P > 0.05). The statistical power of the study is 0.924 at the 0.05 significance level, which is calculated with the power and sample size calculation .To evaluate the association of P = 0.01) and conotruncal defects, which had a 2.18-fold higher  risk in sporadic CHD patients (Table S3). There were no significant associations observed between rs2243890 A>G and other CHD subtypes, including right ventricular outflow tract obstruction (RVOTO) (P = 0.12), left ventricular outflow tract obstruction (LVOTO) (P = 0.34) and patent ductus arteriosus (PDA) (P = 0.41) (Table S3).A further stratified analysis of rs2243890 A>G was performed based on different subtypes of CHDs. The most significant differences in CHD vs. control were observed in an additive genetic model in septal defects, which had a 1.51-fold higher risk in sporadic CHD patients  was located in the promoter region for RCAN1.4 transcript, we hypothesized that the G allele may contribute to the risk of CHD by influencing RCAN1.4 transcription. To test our hypothesis, we evaluated the RCAN1.4 mRNA level in human heart tissue samples from sporadic CHD patients by qRT-PCR. We found that RCAN1.4 mRNA level in human heart samples with heterozygous A/G alleles was almost threefold lower than that of samples with homozygous A/A alleles  probably function as transcriptional repressors.As the minor G allele of rs2243890 decreased RCAN1.4 promoter activity . To assess whether this RCAN1.4E25K mutant plays a role in CHD development, we performed an NFAT promoter-mediated luciferase reporter assay. Results showed that mutant RCAN1.4E25K significantly impaired suppression of NFAT-mediated transcription . About 70% of zebrafishes injected with antisense morpholino-modified oligonucleotides (MO) for zebrafish rcan1a-4 (a zebrafish homolog of human RCAN1.4) , is located in syntenic regions of human chromosome 21. Approximately 40%–60% of DS patients are accompanied by CHD Below is the link to the electronic supplementary material.


Selenium application as sodium selenate was repeatedly shown to have anti-carcinogenic properties by increasing levels of the serine/ threonine protein phosphatase 2A (PP2A) in cancer cells. PP2A has a prominent role in cell development, homeostasis, and in neurons regulates excitability. PP2A, GSK3β and Tau reside together in a complex, which facilitates their interaction and (dys)-function as has been reported for several neurological disorders. In this study we recorded maximum increase in total PP2A at 3 µM sodium selenate in a neuron cell line. In conjunction with these data, whole-cell electrophysiological studies revealed that this concentration had maximum effect on membrane potentials, conductance and currents. Somewhat surprisingly, the catalytically active form, methylated PP2A (mePP2A) was significantly decreased. In close correlation to these data, the phosphorylation state of two substrate proteins, sensitive to PP2A activity, GSK3β and Tau were found to be increased. In summary, our data reveal that sodium selenate enhances PP2A levels, but reduces catalytic activity of PP2A in a dose dependent manner, which fails to reduce Tau and GSK3β phosphorylation under physiological conditions, indicating an alternative route in the rescue of cell pathology in neurological disorders. Selenium is a non-metallic trace element which is incorporated into cells as a component of selenoproteins , includiThe PP2A holoenzyme is composed of a 36 kDa catalytic subunit (PP2Ac) and regulatory and binding subunits PR55B (52 kDa) and PR61B : 52, 52 and 55 kDa) respectively, these latter proteins affect cellular localization and binding to organelles in a site specific manner ,20. Intr2SeO4) that mediates enhanced PP2A activity in neurons remains unclear [The optimum concentration of sodium selenate (Na unclear . In an a unclear . In summ2SeO4) was sufficient to enhance PP2A activity and dephosphorylate specific Tau epitopes in SHSY-5Y cells expressing human tau isoforms [p = 0.046), lower than previously reported [p = 0.002) and 10 µM sodium selenate  SHSH-5Y cells with antibodies against these two proteins, counterstained with DAPI. Here we observed that PP2A and Tau co-localize outside the nucleus in these cells .p = 0.016; p = 0.030; and p = 0.043 respectively; In a series of immunoblots we confirmed that PP2Ac (36 kDa) increased in all treatments normalized to β-actin levels. Surprisingly when mePP2Ac was quantified and normalized to the PP2Ac/β-actin values we recorded a significant decrease at 1, 3 and 5 µM sodium selenate treatment (p = 0.046 and p = 0.040 respectively). For GSK3βY216 we detected significant increases at 3 and 5µM sodium selenate levels (p = 0.009 and p = 0.007), all values were normalized to the total GSK3β and β-actin . Therefore, we investigated the phosphorylation states of GSK3β-ser9 (GSK3βS9), GSK3β-tyr216 (GSK3βY216), under sodium selenate treatment. For GSK3βS9 we recorded significant increase at 1 and 3 µM . For the PHF-1 epitope significant changes were detected for 1, 3 and 5 µM . For this molecule we investigated the phosphorylation states of Tau-ser202 and Tau-ser396 (the latter two are recognized as disease markers AT8-S202 and PHF1-S396) . For theh −70 mV) current injection and established an I-V ratio, that was greatly enhanced for cells treated with 3 µM sodium selenate  = 25.58, p = 0.0007, RM-ANOVA, p = 0.001, p = 0.00057, n = 6 cell/group, t-test). Measurement of the magnitude of the evoked action potential (eAP) when current was injected into cells, again revealed that 3 µM sodium selenate treatment was the optimum concentration in dampening cell excitability—as clearly observed for 2 arbitrarily selected current injection steps 10 and 90 pA. The value for 0 µM sodium selenate at 10 pA was 241 ± 35 mV and at 90 pA, 223 ± 8.2 mV. In comparison for 3 µM sodium selenate, 10 pA was 85 ± 8.9 and 90 pA was 127 ± 8.5 mV , an alternate derivative was found to be more cytotoxic as it interferes with redox reactions in mitochondria [Selenium is a trace element necessary in low doses for cell metabolism. For example, selenium derivatives such as selenocysteine and selenomethionine are incorporated into the production of anti-oxidant proteins GP and TR, which modulate reactive oxide species in cells and have been reported to induce neurogenesis in an AD murine model ,4,53,54.chondria ,48,49. Ochondria ,44. Furtchondria ,55,56,57chondria ,59,60. Tchondria   all material from Thermo Fisher Scientific, . Cells were maintained at 37 °C in a saturated humid atmosphere containing 95% air and 5% CO2. After 5 days in the presence of RA, cells were washed three times with DMEM and incubated 24 h with sodium selenate , dissolved in media to the required concentration .Neuroblastoma SHSY-5Y cells  were prepared and differentiated as described by Encinas et al. ,70. In bIn a 24 well plate, cover slips were pretreated with laminin 10 µg/mL overnight and cells were plated as outlined above. After sodium selenate treatments, cells were rinsed three times with phoshphate buffered saline (PBS) pH 7.4, then fixed in 4% para-formaldehyde for 30 min at room temp (RT) and then subsequently washed three times with TBST (TBS with 0.2% Tween pH 7.4). Fixed cells were then permeabilized using PBST (PBS with 0.2% Triton) for 10 min, washed three times with TBST, and incubated in blocking buffer  with continuous shaking for at least 1 h at RT. After blocking, cells were incubated, with continuous shaking over night at 4 °C, with the desired primary antibodies that have been diluted in the blocking buffer. Afterwards, cells were washed 3 times with TBST, stained with appropriate secondary antibodies (1:2000 dilution) for an hour rocking at RT, were washed three times with TBST. Finally, 0.5 µg/mL Hoechst 33258 dye (“DAPI” Molecular probes) were added to the cells for nuclear staining. After washing with PBS, cover slips were mounted in polyvinyl mounting medium and the cells were viewed and photographed with Axio Ziess fluorescence microscope using 20× and 40× objectives. The number of PP2A positive cells were counted using image J software and PP2A density was normalized to the number of DAPI positive cells. In total for each condition and each replicate a total of six images with an area of 1136.72 × 912.02 µm were counted.Sodium selenate treated and untreated SHSY-5Y cells were trypsinized and suspended in 100 μL staining solution (PBS with 2% FBS and 0.1% sodium azide). Suspended cells were treated with 7AAD  viability dye and incubated for 20 min at 4 °C to discriminate between live and dead cells. Subsequently cells were washed and resuspended in 1 mL flow cytometry staining buffer . Data were acquired with a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Bioscience) and analyzed on BD FACSuite software (BD Biosciences).Total RNA was isolated from tissue samples  and processed for cDNA preparation according to manufacturers’ instructions . qReal-Time PCR was achieved with SYBRgreen mastermix (Thermo Fisher Scientific) with the optimized annealing temperature 60 °C:PP2C-forward-GCCTCTGCGAGAAGGCTAAA and reverse-GAATGGTGATGCGTTCACGG [β-actin-forward-AGAGCTACGAGCTGCCTGAC and reverse-AGCACTGTGTTGGCGTACAG with the latter used as a control. In the study ΔCt was obtained by subtracting the Ct values of β-actin from the Ct values of PP2C, then normalized to the former and plotted as a graph.GTTCACGG  and β-acg. The resulting supernatant was transferred to clean 1.5 mL tubes, from which protein concentrations were quantified in triplicate with BCA colorimetric assay with a BSA standard (Thermo Fisher Scientific BCA assay kit). For immunoblotting. total protein (15 μg) was fractionated through an SDS-PAGE gel (stacking 4–12% resolving) and transferred onto a 0.45 polyvinylidene difluoride membrane . After transfer, membranes were washed in Tris-buffered saline containing 0.1% (v/v) Tween-20, (TBST) pH 7.4, blocked with 5% (w/v) non-fat dry milk in incubated with the relevant primary antibody (1:1000 dilution unless stated), at 4 °C on a rocking platform, overnight. For protein band detection blots were washed 3 × 10 min with TBST pH 7.4 and incubated with horse radish peroxidase (HRP)-linked secondary antibodies  for 1 h at room temperature on a rocking platform, the proteins were detected using enhanced chemiluminescence reagent  and imaged on a ChemDoc system (Biorad). The band densities of the images obtained were then quantified using the basic Quantity One software (Biorad). In studies here β-Actin was used as the loading control. For re-probing, blots were stripped with Restore Western Blot buffer, as per manufacturers’ instructions (Thermo Fisher Scientific) and to confirm no residual activity, blots were probed with secondary—HRP-linked Ab alone and imaged to confirm complete removal of previously tested primary Ab.Total protein from cells was extracted using RIPA Buffer Solution (Thermo Fisher Scientific 89900) with Proteinase (P8465-5ML) and Phosphatase Inhibitor Cocktail 3  added as per manufacturers’ instructions. This cocktail was added to cultured cells at 200 μL/well on ice. Cell lysates were collected in 1.5 mL Eppendorf tubes, centrifuged at 14,000× We evaluated expression of catalytic subunit  PP2A  as well as methylated (Leu 309) PP2Ac . mePP2Ac level was interpreted as an index of PP2Ac activity. The following antibodies GAPDH, β-Actin were purchased from Cell Signaling Technologies . The following antibodies were purchased from Abcam Ltd. : total GSK3β, GSK3β39, GSK3βY216, Total Tau, Tau 231, Tau S396 .+-gluconate, 10 KCl, 10 HEPES, and 2 Mg2+-ATP, and pH 7.20 adjusted with KOH. The extracellular recording solutions were composed of (mM): 140 NaCl, 5.5 KCl, 1 MgSO3·6H2O, 2 CaCl2·2H2O, 15 HEPES, 10 Glucose, and the pH 7.4. The junction potential was ~15 mV and corrected in all experiments. The membrane potentials, under voltage-clamp condition were held at −70 mV and currents were evoked by stepping the membrane potential in series from −40 to +80 mV (10 mV increments) for 50 ms. Action potentials, under current-clamp condition with membrane potential around −70 mV, were evoked by injecting a series of current steps from 10 to 100 pA (in 10 pA increments) for 50 ms, signals were sampled at 50 kHz and filtered at 10 kHz.The SHSY-5Y neurons were visualized through an inverted microscope (Olympus IX53) equipped with an Olympus DP27 camera and a 40× objective . Electrophysiological recordings were with an EPC-10 USB amplifier combined with Patchmaster software . Whole-cell patch clamp recordings were at room temperature (23 °C) on retinoic acid differentiated SHY5Y neurons. Recording electrodes were pulled from a P-97 pipette puller , using borosilicate glass micropipettes  and had a resistance of 5–8 MΩ. Composition of solutions for the recording of membrane properties and evoked action potentials, were composed of (mM): 144 Kt-tests with Welch’s correction, p values noted in the text.Unless otherwise stated all studies were in triplicate with statistical analyses