The intrinsically disordered protein tau associates with microtubules in neurons but aggregates into cross–β amyloid fibrils that propagate in neurodegenerative brains. Different tauopathies have different structures for the rigid fibril core. To understand the molecular basis of tau assembly into different polymorphs, here we use solid-state nuclear magnetic resonance (NMR) spectroscopy to determine the structure of a tau protein that includes all microtubule-binding repeats and a proline-rich domain. This P2R tau assembles into well-ordered filaments when induced by heparin. Two- and three-dimensional NMR spectra indicate that R2 and R3 repeats constitute the rigid β-sheet core of the fibril. Unexpectedly, the amino-terminal half of R2 forms a β-arch at ambient temperature (24°C) but a continuous β-strand at 12°C, which dimerizes with the R2 of another protofilament. This temperature-dependent structure indicates that R2 is conformationally more plastic than the R3 domain. The distinct conformational stabilities of different microtubule-binding repeats give insight into the energy landscape of tau fibril formation. Solid-state NMR data reveal that tau's R2 repeat has high conformational plasticity during amyloid fibril formation. Tau is an intrinsically disordered protein that stabilizes microtubules (MTs) to promote axonal transport . The MT-Although the precise molecular mechanism of tau aggregation is still largely unknown, many structures of aggregated tau have now been determined. Early transmission electron microscopy (TEM) images of paired helical filaments (PHFs) and straight filaments of AD tau showed a fuzzy exterior, which can be removed by pronase to reveal an ordered core , which spans the P2, R1, R2, R3, R4, and R′ domains. Using ssNMR, we have characterized the conformation, dynamics, and three-dimensional (3D) fold of heparin-induced P2R tau fibrils. Unexpectedly, we find that the protein adopts two distinct but related rigid-core conformations at temperatures that are only ~12°C apart. This result indicates an exquisite sensitivity of the conformation of the R2 repeat to environmental conditions, giving insight into the conformational variability of this domain in brain-extracted 4R tau fibrils.Escherichia coli and purified it by heat denaturation, affinity column chromatography, and high-performance liquid chromatography (HPLC) (fig. S1). Fibrils were assembled by mixing tau monomers (0.1 mg/ml) with heparin (0.125 mg/ml) under three conditions: at 37°C with shaking at 300 rpm, at 24°C with rocking at 50 rpm, and at 12°C with shaking at 55 rpm. At 37°C, only amorphous fibrils of less than 50 nm length are observed . Reobserved . Furtherobserved . These o13C cross-polarization (CP) spectra selectively detect immobilized residues, whereas direct-polarization (DP) spectra give approximately quantitative intensities of both mobile and immobile residues. The 12°C fibril shows higher CP intensities than DP intensities compared to the 24°C fibril (fig. S1D), indicating that low-temperature fibril growth produced a larger rigid core. 2D 15N─13C (NC) correlation spectra exhibit about 50 peaks for the 24°C fibril and about 65 peaks for the 12°C fibril (13C─13C (CC) correlation spectra  for 13C and 0.8 to 1.2 ppm for 15N, implying that both fibrils have well-ordered molecular conformations.To characterize the structures of the rigid cores of these P2R tau fibrils, we measured ssNMR spectra. 1D C fibril , and the spectra  show 7 S13C and 15N chemical shifts sequence-specifically using 3D 15N─13C correlation experiments, including NCACX, NCOCX, and CONCA (fig. S2). For the 24°C fibril, we assigned 48 residues, most of which reside between K274 and S320 (table S1) in the R2 and the first half of R3 , which ends at the second 322CGS324 motif. In the 12°C P2R tau fibrils, the chemical shifts delimit four β-strands in the R2 and R3 domains and indicate additional β-strands in the R4 and R′ repeats  indicate that the R2 domain exhibits larger chemical shift differences than the R3 domain.The assigned Cα and Cβ chemical shifts (fig. S5A) allow us to obtain the secondary structures of P2R tau fibrils  and 103 medium-range contacts, in addition to about 500 sequential and intra-residue correlations. We observed correlations between the β1 and β2 strands (I277–S289), the β2 and β4 strands (S289–P312), and the β3 and β4 strands (I297–V308) . These c1-screw symmetric dimer, based on the fact that most brain-derived dimeric tau aggregates exhibit this symmetry , we calculated the structures of both high- and low-temperature P2R tau fibrils using XPLOR-NIH (table S8). For the 12°C fibril, we assumed a 2symmetry  shows a 1KLDL284 . The 301C fibril  shows a 1H magnetization to protein amide protons and detecting the result in 2D 15N─13C correlation spectra (table S9). The relative intensities between the 4-ms and 100-ms water-transferred spectra represent a site-resolved hydration map of the fibril core  fibrils  and is water inaccessible, indicating that this unusual conformation is surrounded by other residues. In comparison, the minor peak (state 2) has a regular 15N chemical shift and is water accessible, suggesting that a small fraction of fibrils contain a disordered and water-exposed 300VPGGG304 loop.Water-edited NMR experiments provide additional restraints to the structures of the two P2R tau fibrils. We measured the water accessibilities by transferring the water bilities , whereas72 in R′ . For the fibrils . G304 ex1-screw symmetry. Although cryo-EM and scanning TEM can give information about the symmetry of amyloid assemblies, the extensive lateral bundling of the 12°C fibrils . This suggests that the ionic environment may influence the structure of this cationic Lys pair. The R3 domain contains four nonconsecutive Lys residues and one Asp. A 311KP312 pair follows the R3 hexapeptide and is the site of a kink in most tau fibril structures. These amino acid sequence differences may contribute to the distinct conformational stabilities of the R3 and R2 domains.These data indicate that different MT-binding repeats of tau have distinct conformational plasticity: The R3 repeat is the most stable amyloidogenic repeat, followed by R2. The R3 strand-turn-strand conformation is largely invariant in both P2R tau fibrils and full-length 0N4R tau (table S5 and fig. S7). In contrast, the N-terminal half of R2 is structurally plastic and can bend onto itself like a hairpin or straighten into a β strand that dimerizes. In full-length 0N4R tau, the same R2 segment shows intermediate disorder, with Although the R2 repeat is conformationally more plastic than R3, we observed a predominant set of chemical shifts at both temperatures, indicating that the two fibril structures do not coexist. Thus, there is a substantial energy barrier between the three-layered structure and the four-layered dimer structure. This high energy barrier is consistent with current understanding of the free energy landscapes of amyloid formation. Coarse-grained molecular dynamics simulations of Alzheimer's β amyloid (Aβ) fibrils found a small number of distinct minima that are separated by high energy barriers  studied here is similar to K32 tau (198 to 400), originally designed to investigate tau interactions with MTs , a tobacco etch virus (TEV) cleavage site (6 residues), and residues 198 to 399 of tau. Following TEV cleavage, a Gly residue remains before the N-terminal S198 in the final purified protein. This gene was cloned into a pET-28a vector and transfected into E. coli BL21(DE3) competent cells (New England Biolabs). A starter culture was grown in 50 ml of LB medium containing kanamycin (50 μg/ml). After overnight growth at 37°C with shaking at 220 rpm, the 50-ml overnight culture was used to inoculate 1 liter of LB medium containing kanamycin (50 μg/ml). Cells were grown at 37°C and 220 rpm until optical density at 600 nm reached 1.0, then the cells were spun down at 1000g and 4°C for 20 min, and the cell pellet was resuspended in 1 liter of minimal medium containing M9 salts, 15NH4Cl (1 g/liter), 13C-labeled glucose (2 g/liter), 1 mM MgSO4, 0.1 mM CaCl2, kanamycin (50 mg/ml), vitamin, and mineral supplements. Cells were grown at 37°C under shaking at 220 rpm for 2 hours, and, then, protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). At this point, another 13C-labeled glucose (1 g/liter) was added to the medium to give a total of 13C-labeled glucose (3 g/liter). Expression proceeded for 18 hours at 30°C under shaking at 180 rpm. Expression of 0N4R tau was induced with 0.5 mM IPTG and lasted 5 hours at 37°C with shaking at 180 rpm.The gene encoding P2R tau contains an N-terminal His6-thrombin-TEV-P2R tau fusion protein was purified using Ni2+ affinity column chromatography. Then, the His6-thrombin-TEV tag was removed using TEV protease, and the native P2R tau was purified by reverse-phase HPLC. In detail, cells were spun down at 6000g and 4°C for 15 min. The pellet was resuspended in 40 ml of a lysis buffer containing 1× phosphate-buffered saline (PBS) (Sigma-Aldrich) , 2 mM dithiothreitol (DTT), 0.05 mM phenylmethylsulfonyl fluoride (PMSF), and one tablet of cOmplete protease inhibitor cocktail (Roche). The cells were lysed by sonication on ice . The lysate was boiled for 10 min and then spun at 20,000g for 60 min to remove cell debris and aggregates of heat-sensitive cellular proteins. The supernatant containing the fusion protein was loaded on 5 ml of Profinity IMAC resin that had been charged with Ni2+  and washed with the binding buffer . After loading, the resin was washed with 20 column volumes of binding buffer, and, then, the proteins were eluted from the column using 5 column volumes of the elution buffer . The eluted fraction was loaded onto a desalting column  to exchange buffer for the TEV cleavage buffer . The solution was then diluted to the fusion protein (~0.5 mg/ml) to prevent aggregation during cleavage. Subsequently, TEV protease was added at a ratio of 3:100 (w:w) with respect to the fusion protein, and the cleavage reaction proceeded for 16 hours at 4°C with gentle rocking. After cleavage, 2 M guanidinium chloride was added to the solution and then loaded onto 5 ml of Ni2+-charged resin that had been prewashed with the binding buffer. The flow-through containing the P2R tau protein was collected and then purified on a reverse-phase HPLC using a preparative C3 column  and an acetonitrile gradient of 5 to 95% in 35 min. The eluted P2R tau fractions were >95% pure as determined by SDS–polyacrylamide gel electrophoresis with Coomassie blue staining. The fractions were pooled and lyophilized to give the P2R tau powder. The yield of the expression and purification was ~15 mg of protein per liter of M9 culture.The His−1 cm−1) due to its lack of Trp and the small number of other aromatic residues, which makes absorbance measurements unreliable. -labeled P2R tau powder was dissolved in 1× PBS buffer containing 2 mM DTT to reach a concentration of 2 mg/ml and then bath-sonicated for 5 min to remove small aggregates. This monomer solution was transferred to 50-ml Falcon tubes and diluted to 0.1 mg/ml (4.7-μM monomer), and, then, heparin was added to a final concentration of 0.125 mg/ml .The amount of P2R tau was measured gravimetrically with an estimated uncertainty of 15%. We used this method of quantification because P2R tau has a small extinction coefficient (2980 MThree temperature and agitation conditions were used for fibril growth: (i) 37°C with shaking at 300 rpm (2.54-cm orbit); (ii) 24°C with rocking at 50 rpm; and (iii) 12°C with shaking at 55 rpm (2.54-cm orbit). In conditions (i) and (iii), we strapped 10 Falcon tubes (50-ml) each containing 25 ml of solution horizontally on an incubator platform  to induce a “sloshing” movement of the solution along the tube axis. In condition (2), the 10 Falcon tubes (50-ml) were strapped horizontally on a rocker platform  to induce a similar sloshing movement of the solution. For conditions (ii) and (iii), the rocking and shaking rates were chosen to cause similar amounts of agitation. For condition (i), the high temperature and fast shaking were designed to mimic the previously used conditions to produce full-length 0N4R tau (g for 1 hour and 4°C using a fixed-angle rotor (Beckman TLA55). Hydrated pellets were spun into 3.2-mm Revolution NMR pencil rotors using a funnel made of pipette tips and a swinging-bucket centrifuge at 4000g for 5 min. The rotors contained ~36 mg of hydrated mass in which ~20 mg was estimated to be P2R tau fibrils.The solutions were incubated for 3 days but became cloudy after about 1 day. The samples prepared at 12° and 24°C were concentrated by ultracentrifugation at ~112,000Unconcentrated P2R tau fibril solutions were adsorbed onto freshly glow-discharged, 200-mesh formvar/carbon-coated copper grids (Ted Pella), extensively washed with water to remove any trace of PBS buffer, and stained with 0.7% (w/v) uranyl formate for 15 s. TEM images were taken on an FEI Tecnai T12 electron microscope.1H chemical shift, which was calibrated using DSS (sodium trimethylsilylpropanesulfonate)  spectrometer at the Francis Bitter Magnet Laboratory using a Black Fox 3.2-mm HCN MAS probe. Sample temperatures were measured using the water 13C MAS spectra were measured using different polarization transfer methods to detect residues with different mobilities. A 1H─13C CP spectrum measured with a contact time of 70 μs was used to detect the rigid residues, while a CP spectrum measured with a contact time of 800 μs was used to observe both rigid and semirigid residues. Highly mobile residues were detected using 1D and 2D 1H─13C Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) spectra, as well as 1H─15N INEPT spectra.1D 15N and 13C chemical shifts of the rigid core, we measured 2D 13C─13C and 15N─13C dipolar correlation spectra and 3D NCACX, NCOCX, and CONCA correlation spectra. To obtain long-range distance constraints for structure calculation, we conducted 3D CCC experiments. These 2D and 3D dipolar correlation experiments required a total of 4 weeks of signal-averaging per sample. 15N─13C polarization transfer used the SPECIFIC cross-polarization (SPEC-CP) sequence (1H─13C CP spectrum. 13C spin diffusion was achieved using the Combined-Driven (CORD) sequence  torsion angles using the TALOS-N software  to enhance spectral resolution or GM apodization (LB = −20 Hz and GB = 0.05) to enhance sensitivity. Spectra were first processed in the Topspin software and then exported to the CCPNMR software for chemical shift assignment  and the 100-ms spectrum (S0) give information about the relative water accessibilities of the residues. Uncertainties in the S/S0 values were propagated from the signal-to-noise ratios of 1D cross sections of the 2D spectra, using empty regions of the spectra as the noise.To investigate the water accessibility of the fibril core, we measured water-edited 2D N-Cα correlation spectra. The experiment starts with a water-selective TCβ − ΔδCα ≡  − , is used to define the β strand segments. Positive ΔδCβ − ΔδCα values denote β strand segments, while negative values indicate α-helical segments. Chemical shift differences between the 24° and 12°C P2R tau fibrils are calculated as root mean square deviations of the Cα, Cβ, and CO chemical shifts.The 2D and 3D dipolar correlation spectra were sequential assigned following standard protocols. 2D CC, 2D NCα, and 3D NCACX spectra were first analyzed to identify the residue types of all rigid and semirigid residues, and, then, the Cα and CO chemical shifts were used in conjunction with 3D NCOCX and CONCA spectra for sequential assignment. A composite metric of the secondary chemical shift difference between Cβ and Cα, Δδz axis with a 20-Å spacing between monomers. The noncrystallographic symmetry potential PostDiffPot was used to restrain the monomers to be structurally identical. Fictitious Nuclear Overhauser Effects (NOEs) between the Cα atoms of the same residue of adjacent chains were used and set to 4.8 ± 0.1 Å. An artificial residual dipolar coupling (rdcPot) potential was applied to the Cα atoms in adjacent chains to align the monomers along the z axis. A total of 42 pairs of  angles were inputted (i − j > 1) and 36 long-range correlations (i − j ≥ 4) were used as intramolecular distance constraints and were given an upper bound of 8.5 Å. NOE potentials were always applied with the “soft” flag.The rigid-core structures of the two P2R tau fibrils were determined using XPLOR-NIH hosted on the NMRBox computing platform  potential was applied to the Cα atoms in adjacent chains to align the monomers along the z axis. An additional 21 screw symmetry was applied with DistSymmPot potential to restrain the monomers in the two stacks, which is so far the main symmetry observed for all brain-extracted dimeric tau fibrils (i − j ≥ 4) were used as intramolecular distance constraints, among which three long-range constraints were explicitly set as dimer interface contacts. NOE potentials were always applied with the soft flag.For the 12°C P2R fibrils, 5 × 2 monomers of the protein (residues 264 to 326) were first aligned along the Structure calculation in XPLOR-NIH consisted of minimization, simulated annealing, and refinement of the protein. In the minimization step, three extended fibrils made of 5 or 10 copies of P2R tau each were minimized in torsion angle space, first for 2000 steps with only standard bond length, bond angle, and improper angle potentials, then for 5000 steps with these constraints, as well as nonbonded repulsion, PosDiffPot, and the Cα-Cα artificial distance restraints. In the simulated annealing step, 1000 independent XPLOR-NIH runs were performed using high-temperature torsion angle dynamics at 5000 K for 10,000 steps, followed by simulated annealing to 20 K in 20-K decrements with 500 steps of torsion angle dynamics at each temperature. Final energy minimizations were performed in torsion angle and Cartesian coordinates. During all calculations, standard XPLOR-NIH BOND, ANGL, IMPR parameters were used to set the bond lengths and bond angles.


Thrombocytopenia is one of the symptoms of many virus infections which is the “hallmark” in the case of dengue virus. In this study, we show the differential localization of existing two forms of dengue virus protease, i.e., NS2BNS3 to the nucleus and NS3 to the nucleus and mitochondria. We also report a nuclear transcription factor, erythroid differentiation regulatory factor 1 (EDRF1), as the substrate for this protease. EDRF1 regulates the expression and activity of GATA1, which in turn controls spectrin synthesis. Both GATA1 and spectrins are required for platelet formation. On the other hand, we found that the mitochondrial activities will be damaged by NS3 localization which cleaves GrpEL1, a co-chaperone of mitochondrial Hsp70. Levels of both EDRF1 and GrpEL1 were found to deteriorate in dengue virus-infected clinical samples. Hence, we conclude that NS2BNS3-mediated EDRF1 cleavage and the NS3-led mitochondrial dysfunction account for thrombocytopenia.  •Dengue virus protease exists in two forms i.e. non-structural protein-3 (NS3) and NS2BNS3•NS3 localizes to both mitochondria and nucleus whereas NS2BNS3 localizes to nucleus only•NS3 cleaves GrpEL1 of mitochondrial matrix, and NS2BNS3 cleaves EDRF1 of the nucleus•EDRF1 cleavage by NS2BNS3 and mitochondrial dysfunction by NS3 account for thrombocytopenia Molecular biology; Virology; Cell biology The World Health Organization (WHO) reported an 8-fold increase in dengue cases in the past two decades with approximately 5.2 million cases in the year 2019 alone.flaviviridae. There are four distinct serotypes (DENV1-4) that have emerged from sylvatic strains in the forests of Southeast Asia. DENV is an enveloped, single-stranded positive-sense RNA virus. The RNA genome consists of approximately 10,700 nucleotides and codes nearly 3,411 amino acids (a.a) long precursor polyprotein yielding three structural proteins (Capsid (C), precursor Membrane (preM), and Envelope (E) and seven non-structural (NS) proteins .,,,,,,,,,,,,in vitro, ex vivo, and in vivo models have been studied, the mechanism behind the reduction in platelet cell number causing thrombocytopenia has not yet been clearly understood.,,Dengue virus (DENV) is a flavivirus of family ,,Virus-mitochondria interaction is being revealed in several reports suggesting mitochondrial dysfunction.,,,,Ex vivo and in vivo  analyses clearly supported the above observation. EDRF1, in turn, regulates another two factors (GATA1 and Spectrins) of the cells (play a key role in proplatelet and platelet formation) which are also downregulated in protease-transfected or virus-infected conditions. Dengue virus-infected K562 cells (megakaryocyte derived) show reduced cell number as the number of days progressed. Mitochondrial dysfunction of the protease-transfected cells was observed which also accounts for the reduction in cell number. Therefore, we felt that this study gave the molecular-level explanation for thrombocytopenia and facilitates focused therapeutic development.Thrombocytopenia, i.e., reduced platelets is a health disorder that occurs during different ill health conditions including many viral infections which led to the death of millions of people across the globe. Dengue virus infections are known from ancient times which cause “thrombocytopenia” as the primary symptom. Platelet activation, their clearance, and plasma leakage are proposed as the reasons for the platelet reduction in dengue virus patients.in silico method, NLS sequences were identified in both NS2BNS3 (594 a.a) and NS3pro-helicase (464 a.a). In NS2B (130 a.a), three basic amino acid residues 1471KKKQR1475 at position 1471–1475 were identified which are similar to classical monopartite NLS stretch that consists of basic amino acid residues with motif K (K/R) X (K/R). In NS3 sequence, two stretches of bipartite signal (2–3 positively charged amino acid followed by 9–12 linker sequence containing proline residue) were identified at positions (a.a 1656–1716 and 1839–1856) with a score of 4–4.1 , stained with Hoechst stain and analyzed for the localizations. The data suggested GFP and the MitoRFP expression are not merged suggesting no localization of NS2BNS3 to the mitochondria B ii. It With the cell lysates of the above experiments, we have carried out the western blotting analysis (anti-NS2BNS3pro antibody) to verify the presence of protease forms that localize to nucleus or mitochondria or both. The immunoblotting results were found to be consistent with the above localization experiments suggesting that the NS3pro-helicase (75 kDa) localized to both cytoplasm (mitochondria) and nucleus E ii, whein vitro nickel-nitrilotriacetic acid (Ni-NTA) pull-down assay with purified NS2BNS3pro incubating with K562 whole cell lysate  at different amino acid sequence positions of EDRF1  C. In ordd models A. It wasd models B. Thus, The cell extracts co-expressed with both EDRF1 and protease were resolved on 10% SDS PAGE and transferred onto the hydrophilic polyvinylidene fluoride (PVDF) membrane. Western blot analysis was done to detect the levels of EDRF1 using anti-Myc tag antibody. The blot showed no band in co-transfected vectors as the Myc tag is only 1.2 kDa  H lane 1.To confirm the expression of NS2BNS3pro in the above experiment, we have checked the expression of protease using anti-GFP antibody (pEGFPN1-NS2BNS3pro) after stripping the same membrane. It was observed that, in co-transfected vectors and pEGFP-N1 vector alone, GFP was expressed J lane 16,,,The GATA1 expression and activity were reported to be regulated by EDRF1.To further confirm the above result, we extended our study using the K562 virus-infected cell lysates (7 days post infection) as mentioned in Total forty-four albumin out clinical samples with different grades of infections, i.e., DF, DHF, and DSS were included for the analysis . Other fNS3 was shown to target the mitochondrial matrix and cleave GrpEL1, a co-chaperone of mitochondrial Hsp-70 (mtHsp-70), in our previous study.Oxygen consumption rate (OCR) is a measure of the total cellular respiration in cells that meet the cellular ATP demand of the cell under baseline conditions. Basal levels of OCR in NS3pro-helicase-transfected cells were found to be decreased compared to vector-transfected cells which suggested low mitochondrial respiration in transfected cells C i.FigurOn the addition of oligomycin, F0/F1 ATPase (complex V) shuts off, which relates to the activity of mitochondria generating the ATP linked to basal respiration. It was observed that in presence of oligomycin, the ATP production was compromised in NS3pro-helicase-transfected cells when compared to vector C ii. A dOn the addition of Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (uncoupler), the movement of protons (H+ ions) will be allowed. This phenomenon leads to sudden increase in maximal respiration indicating high substrate availability and good integrity of the ETC as observed in vector-transfected cells. However, there was no increment in maximal respiration in NS3-pro helicase-transfected HepG2 cells C iii indIt is a measure of the difference between maximal respiration and basal respiration and represents the cell fitness and the cell response due to high energy demand. In NS3pro-helicase-transfected cells, maximal respiration was found to be low compared to vector; thus there was low spare respiratory capacity compared to the vector. Low respiratory capacity indicates low fitness of the cell Civ.Basal respiration that is not linked to ATP production indicates the proton leak. There was no significant change in proton leak due to low energy demand or low substrate availability in transfected cells C v.Addition of antimycin and rotenone inhibits complex III leading to low cellular respiration. Non-mitochondrial oxygen consumption is the measure of the cells recovering their energy demand through other cellular processes. In the NS3pro-helicase-transfected cells, there was no increase in non-mitochondrial OCR as compared to the vector indicating the damage to mitochondria C vi.In order to analyze the effect of virus infection on K562 cells (megakaryocyte-derived cell line), we performed a cell counting method to analyze the cell number during virus infection. For this purpose, K562 cells with dengue-infected or uninfected were analyzed day wise  for morphological changes. Fourth day post infection onwards, visible cell morphological changes  occurred. Total cell count starts decreasing after day 3. The rounded cell morphology of K562 cells was changed, a visible reduction in cell size on seventh day was observed, and the changes were imaged under an inverted bright-field microscope (magnification at 10X)  A and 7B,,Dengue virus infections occupied the top of the list of emerging infectious diseases at present, but there is no specific drug or vaccine until now. All four serotypes cause similar disease manifestations individually or in combination with other serotypes. Flaviviruses target many host cellular proteins for efficient viral replication. The proteases of dengue and other flaviviruses  are known to cleave self-polyprotein and host proteins.In order to identify the substrates of NS2BNS3/NS3, if any, from the nucleus, we have carried out the pull-down experiment consisting of total cell extracts with NS2BNS3pro. The co-eluted protein bands were identified by mass spectrometry analysis and were found to be EDRF1 F and 4G.ex vivo to in vivo  conditions , and its activity is modulated by the EDRF1 protein. EDRF1 has been reported to play a major role in transcriptional regulation and expression of GATA1 and globin gene expression, a key factor in megakaryopoiesis.nditions  and S4. ,Reports suggest defective mitochondrial quality and dysfunctioning during viral infections including dengue virus.This study was approved by the Institutional Ethics Committee, University of Hyderabad (UH/IEC/2021/1).The present study carried out is completely around the dengue virus protease and its effect on cell homeostasis. Availability of a specific anti-protease molecule would have shed light more clearly on the observations. But the reported two anti-protease drugs quercetin and Nordihydroguaiaretic acid (NDGA) are highly cell toxic and hence were not used in this study. Hence the availability of a specific anti-protease molecule could be considered as the limitation in the present study.mvrsl@uohyd.ernet.in).Further information and reasonable requests for resources and reagents should be directed to the lead contact, Dr. Musturi Venkataramana , Vero cells (monkey kidney cells), Hepatocellular carcinoma (HepG2) and Megakaryocyte derived cells (K562) were obtained from National Centre for Cell Science [NCCS], Pune, India. HEK, HepG2 and Vero cells were maintained in Dulbecco’s Modified Eagle’s medium . K562 cells were maintained in RPMI media  at 37°C in a humidified incubator with 5% CO2. Both DMEM and RPMI were supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic containing (vol/vol) penicillin and streptomycin.KX618706.1, Protein id. ASD49618.1. NLS in both the sequences were determined using the cNLS mapper software with a cutoff score of 0.4 and also identified the residues that show the monopartite and bipartite NLS sequences based on classical nuclear localization signal.To identify the nuclear localization signal in NS2BNS3 and NS3, the amino acid sequences were retrieved from the NCBI data base of the in-house characterized DENV1 with Accession no. GGATCCGCTGATTTATCATTGGAGAAA and NS2B Reverse – 5 CCCGCCTCCACCACTACCTCCGCCCCCGAGCGTGTCATCTCTCTCTTCAT 3’ primers. NS3pro was amplified using NS3 Forward-5’GGGGGCGGAGGTAGTGGTGGAGGCGGGAGAGCAGTTCTTGATGATGGTA and NS3 Reverse 5’ATCGAGAATTCTTACCTAAACACCTCGTCCTCAATC3’ primers. The obtained gene products were used as templates with external primers (the above forward primer of NS2B and the reverse primer of NS3) for overlap extension PCR to generate NS2B-G4-S-G4-NS3pro. This amplified DNA was digested with ECoRI and BamHI enzymes and then ligated to the pRSET-A vector. The clone was confirmed by sequencing. In order to develop pEGFP-N1 NS2BNS3pro, the above pRSET-A NS2BNS3pro was used as a template with forward 5’ TATGCTCGAGATGGCTGATTTATCATTGGAC 3’ and reverse 5’ TATCGGATCCGTAAACACCTCGTCCTC 3’ primers. The amplified fragment (696 bp) and pEGFP-N1 vector were digested with XhoI/BamHI enzymes and gel purified by gel extraction kit (Thermofisher Scientific). The digested products were ligated using T4 ligase enzyme. Ligated product was transformed into E. Coli. DH5α competent cells and the isolated plasmid was confirmed with restriction digestion using XhoI/BamHI enzymes. The positive clone pEGFP-N1 NS2BNS3pro was further confirmed by sequencing. pEGFP-N1 NS3pro-helicase (464a.a) construct was also developed during our previous study.CTCGAGATGGGATGGTATCTATAGA3’ and reverse 5’ GGATGTAGGTCCATTATTGTTAGGT 3’ primers. Helicase was amplified using forward F 5’ACATCCAGGATCAGGAAAAACA 3’ and reverse 5’TATCGGATCCCCCATGTAAATATACTGG 3’ primers. The reaction was performed in a final volume of 25μl containing 0.5 μl cDNA template, 2.5 μl reaction buffer, 0.5μl of 10mM dNTPs, 0.25U of Taq polymerase enzyme, 0.5 μl of each of 10 μM forward and reverse primers. Cycling conditions were followed with an initial denaturation step at 95°C for 4 minutes, followed by 35 cycles of 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 1 minute, and final extension at 72°C for 10 minutes. The final products were electrophoresed on 1% agarose gel and purified by gel extraction method. Further overlapping extension PCR was carried to generate NS3pro-helicase (464a.a) using forward primer of NS3 protease containing XhoI restriction site at 5’ end and reverse primer of helicase containing BamHI restriction site at 3’ end as mentioned above. The plasmid construct, NS3pro-helicase consists of 185 amino acids protease domain of NS3 and 283 amino acids of helicase domain. The obtained clone was ligated into pJET 1.2 vector and further subcloned into pEGFP-N1 vector using XhoI/BamHI restriction sites to obtain pEGFP-N1 NS3pro-helicase (464a.a).All the constructs were generated from the in-house characterized DENV1 serotype . pRSET-A NS2BNS3pro (46a.a+185a.a.) clone was generated earlier in our previous study.E. coli. DH5α competent cells. Plasmid was isolated, and insert was confirmed by restriction digestion. To sub-clone pJET 1.2 NS2BNS3 into pEGFP-N1 vector, pJET 1.2 NS2BNS3 and pEGFP-N1 vector were subjected to XhoI/HindIII restriction enzyme digestion. 1.8 kbp digested product from pJET1.2 clone was obtained and further ligated to linearized pEGFP-N1 vector. The ligated recombinant was transformed into E. coli. DH5α competent cells. Plasmid was isolated, and insert was confirmed by restriction digestion and further by sequencing. To clone NS2BNS3pro (S135A) mutant (130+185a.a), NS3pro (S135A) was generated by site directed mutagenesis. The dengue virus NS3 protease inactive mutant was generated by amino acid substitution Ser135 in catalytic triad with Ala using the primers Forward, 5’-TTTTAAACCCGGCACAGCTGGATCTCCC 3’ and Reverse, 5’-TCACGATGGGAGATCCAGCTGTGCCGG 3’. Above amplified NS2B (130a.a) and NS3pro (S135A) (185a.a) were overlapped with overlapping extension PCR using forward 5’ GCCCCTCAATGAAGGAATTATGG 3’ of NS2B at 5’ end and reverse 5’ GGATGTAGGTCCATTATTGTTAGGT 3’ primers of NS3 at 3’ end as described above. The obtained fusion fragment was electrophoresed on 0.8% agarose gel and purified by gel extraction method. NS2BNS3pro (S135A) mutant was ligated to pJET 1.2 blunt end vector using T4 ligase and clone was confirmed using XhoI/HindIII restriction digestion. The digested NS2BNS3pro (S135A) mutant was further subcloned to pEGFP-N1 vector as described above. NS2BNS3 (594 a.a) and NS3pro-helicase (464a.a) are the naturally existing forms of dengue virus infections.In order to develop pEGFP-N1 NS2BNS3 (130+464=594 a.a), NS2B fragment was amplified using NS2B forward 5’ GCCCCTCAATGAAGGAATTATGG 3’ and NS2B reverse 5’ TGATCTCTGTTTCTTTTTCTGCCA 3’ primers. The reaction was performed in a final volume of 25μl containing 0.5μl of cDNA template, 2.5μl reaction buffer, 0.5μl of 10mM dNTP mix, 0.25U of Taq polymerase enzyme, 0.5μl of each 10 μM forward and reverse primers. The pTZ57RT-NS2BTA construct that existed in lab was used as a template. Cycling conditions were followed with an initial denaturation step at 95°C for 4 minutes, followed by 35 cycles of 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 1 minute, and final extension at 72°C for 10 minutes. The final products were electrophoresed on 1% agarose gel and purified by gel extraction method and used as templates for overlapping extension PCR. The reaction mixture was prepared by adding the templates, NS2B and NS3pro-helicase (100ng each), 1X buffer, 10mM dNTP and Q5 polymerase. Two step overlapping extension PCR was carried out to join NS2B at the 5’ end of NS3pro-helicase: 94°C for 4 minutes,  10 cycles followed by 72°C for 15 minutes as a final extension. Later, external primers (NS2B forward & helicase reverse) were added and followed by 35 cycles of second round of PCR, 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 1.4 minutes, and final extension at 72°C for 10 minutes. The obtained fusion fragment was electrophoresed on 0.8% agarose gel and purified by gel extraction method. The overlapping extension PCR amplified product of NS2BNS3 (594 a.a) was ligated into pJET 1.2 using T4 DNA ligase. The ligated product was transformed into GAATTCGCCATGGCTGATTTGTCTACAGACTT 3’ and reverse 5’ TATGCTCGAGCTGAACGGCATTGCTGCT 3’ primers. The primers were designed based on the EDRF1 sequence available with accession no NP_001189367.1. The PCR mix contains 10X buffer, 10mM dNTP, forward and reverse primers (10μM each), cDNA (100ng), Taq Polymerase (0.25U) and water. Cycling conditions were followed with an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 58°C for 30 seconds, 72°C for 1.5 minutes, and final extension at 72°C for 10 minutes. The amplified fragment was run on 1% agarose gel and 1.3 kbp amplified product was obtained. To further clone the amplified fragment in pcDNA3.1 c-myc vector, both the vector and insert were digested with restriction sites XhoI/ECoRI, ligated and transformed into E. Coli. DH5α competent cells. The recombinant plasmid was isolated and further confirmed with restriction digestion and sequencing.From K562 cell pellet, total RNA was isolated using Trizol as per manufacturer’s protocol. For cDNA synthesis, total isolated RNA was mixed with oligo(dT) primers in sterile RNase free water and reaction was followed up with mixing: total RNA (1μg), oligo(dT) random primer mix (0.5μg/μl), 10 mM dNTP, and RNase free water. The RNA was denatured for 5 minutes at 65°C. Then 10X AMV buffer, AMV Reverse Transcriptase (10U/μl), RNase inhibitor (40U) and RNase free water were added. Total RNA was reverse transcribed using the reaction cycle: 45°C for 50 minutes, 80°C for 5 minutes. The cDNA generated was used as template for amplification of EDRF1 with forward 5’ TATC5 cells were seeded in 6-well plate. Transfection was performed with the above mentioned constructs using Lipofectamine 2000 reagent as per manufacture’s protocol in ratio (1.5μg plasmid and 3μl Lipofectamine). After 28 hours the cells were harvested, washed with 1X PBS and fixed using 4% paraformaldehyde for 20 minutes. Then the cells were permeablized with 0.25% Triton-X 100 in 1X PBS for 5 minutes, washed with 1X PBS followed by nuclear staining with Hoechst 33342 stain (Molecular probes). The cells washed and resuspended in 1X PBS and Glycerol (1:1) and mounted on to glass slide with a coverslip. The cells were analyzed using laser scanning confocal microscope (Carl Zeiss) at 20X and 100X magnifications. The images were merged in order to observe the co-localizations. The localization color (green/blue) intensity profiles were prepared using NIS Elements AR software program to analyze the GFP (green peak) in nucleus along with Hoechst stain (blue). The fluorescence percentage was calculated using . p- values indicated p<0.0001, ∗∗∗∗ = significant, ns = non-significant.Transient transfections were performed using the constructs pEGFP-N1 vector, pEGFP-N1 NS2BNS3pro (46a.a+185a.a), pEGFP-N1 NS2BNS3pro (S135A) mutant (130 a.a + 185 a.a) and pEGFP-N1 NS2BNS3 (130 a.a + 464 a.a) in K562 cells. Briefly, one day before transfection, approximately 10In order to analyze the cross localizations of NS2BNS3 (in mitochondria) and NS3pro-helicase (in nucleus), we have used MitoRFP vector and Hoechst satin for both the constructs. In this direction, transient transfections were performed in HEK cells using the constructs pEGFP-N1 vector, pEGFP-N1 NS2BNS3 (594 a.a) and pEGFP-N1 NS3pro-helicase (464 a.a) along with MitoRFP vector. HEK cells were seeded onto coverslips in 12 well plate and the plasmid constructs were transfected along with mitoRFP vector (1μg to 2μl Lipofectamine). After 48 hours, the cells were washed with 1X PBS and fixed using 4% paraformaldehyde for 20 minutes and permeablized with 0.25% Triton-X 100 in 1X PBS for 5 minutes, followed up with staining with Hoechst stain (nucleus) and mounting the coverslip with glycerol on to glass slide. The cells were analyzed using laser scanning confocal microscope at 60X magnification and the images were merged for the analysis. The localization intensity profiles were prepared using NIS Elements AR software program. The fluorescence percentage was calculated as above.Similar transfections were performed  and pEGFP-N1 NS3pro-helicase (464 a.a)) as described above in HEK cells. For preparing whole cell lysates, cells were harvested and washed with 1XPBS by centrifuging at 3000rpm for 5 minutes. The cell pellet was resuspended in RIPA buffer, incubated on ice and vortexed 2-3 times in intervals for 15 minutes. The solubilized lysate was centrifuged at 14000rpm for 15 minutes and the supernatant was collected as whole cell lysate. For preparing cytoplasmic and nuclear fractions, cells were washed with ice-cold 1X PBS, mildly homogenized in hypotonic buffer  and incubated for 10 minutes. The homogenized lysate was centrifuged at 2000g at 4°C for 10 minutes. The supernatant collected as cytoplasmic fraction and the pellet was used for nuclear extraction. The pellet was further washed twice with 1XPBS and resuspended in RIPA buffer. The resuspended pellet was incubated for 30 minutes on ice and vortexed at 5 minute intervals. The completely dissolved pellet was allowed for centrifugation at 20,000g for 15 minutes. The supernatant was collected as a nuclear fraction. The lysates were quantified using Bradford reagent and resolved on 10%SDS PAGE and immunoblotted with NS2BNS3pro antibody (1:1000), anti-GFP antibody (1:1000) and actin (1:2500).K562 cells were cultured in 100mm dishes. The cells were harvested and lysed with RIPA buffer. The lysate was quantified by Bradford reagent (Bio-Rad) and 250-300μg protein (500μl) lysate was used for pulldown. pRSET-A NS2BNS3pro purified protein (200μg) was allowed for binding to Ni-NTA His beads for 3 hours with gentle rocking and centrifuged for 1 minute at 4°C to remove the unbound protein. K562 cell lysate incubated with the protein bead complex overnight with gentle rocking at 4°C. The complex was centrifuged for 1 minute and unbound proteins were collected as flow through (FT), followed by washes of 10-50mM imidazole (500μl each). The interacting proteins were eluted from washed bead complex using imidazole (100-300mM).Elutes, FT washes and the bead complex were mixed with 4X Laemmli buffer and boiled for 10 minutes, centrifuged for 1 minute and resolved on 10% SDS PAGE. A similar procedure was followed up with Ni-NTA beads bound to K562 cell extract without purified protein (control). The gel containing the bands were eluted and followed up by MALDI TOFF mass spectrometry analysis . Further, the above-mentioned flow through, washes and elutes were resolved on 10% SDS PAGE, transferred onto PVDF membrane and probed with anti-EDRF1 antibody (1:3000). The anti-rabbit secondary HRP conjugated antibody was used (1:10000) and the developed image was recorded .Q3B7T1.1, was retrieved from NCBI data base. Protease cleavage sites were identified by using ProP-1.0 Server-DTU Health Tech. Further, the protease and EDRF1 structures were superimposed using auto docking tool to identify the cleavage sites for the protease catalytic triad.Full length EDRF1 protein sequence accession no: HEK cells were seeded and allowed to grow up to 80% confluency. pEGFP-N1 NS2BNS3pro (46 a.a + 185 a.a) was transiently co-transfected with pcDNA3.1 c-myc EDRF1. Transfection was done using Lipofectamine 2000 reagent (1:3 ratios) as per the manufacturer’s protocol. After 48 hours, cells were harvested and proceeded for western blotting and the cleavage was analyzed using anti-Myc tag (1:1000) and anti-GFP antibodies (1:1000). Secondary HRP-conjugated anti-mouse and anti-rabbit antibodies (1:10000), respectively were used.5 cells/ml in 6 well plates prior to the day of transfection in RPMI growth media with 10% FBS without antibiotic and incubated at 37°C with 5% CO2. After 12 hours, cells were transfected with pEGFP-N1 vector, pEGFP-N1 NS2BNS3pro, pEGFP-N1 NS2BNS3pro (S135A) mutant, pEGFP-N1 NS2BNS3 (1-5μg/μl) and allowed to grow for 24 hours. Cells were analyzed for GFP expression using fluorescence microscope. Then the cells were harvested and cell extracts prepared, resolved on 10% SDS PAGE and probed with anti-GFP (1:1000) or anti-NS2BNS3pro (1:5000) or anti-EDRF1 (1:3000) or anti-GATA1 (1:5000) or anti-alpha1 spectrin (1:2500) antibodies . The secondary anti-rabbit HRP-conjugated antibody (1:10000) was used and anti-actin (1:2500) was used and probed with HRP-conjugated anti-mouse secondary antibody (1:10000). The blot was developed and imaged using Chemidoc (Bio-Rad).Cells were seeded at conc. of 5x106 cells. On the day of infection, cells were harvested and counted using a hemocytometer. Suspension cells were seeded again in 60mm  were classified as DF, DHF and DSS as per WHO guidelines . The sam2. Next day, sensory cartridge is equilibrated with XFp Calibrant solution for 2 hours. Later, the sensory cartridge was filled with three drugs  as per protocol and XFp cell mitostress assay was performed using Seahorse XFp Analyzer with the in-built software program.,Transient transfection was performed in HepG2 cells with plasmid constructs pEGFP-N1 vector and pEGFP-N1 NS3pro-helicase as per manufacturer’s protocol. 28 hours post transfection 7000-8000/well cells were seeded in Agilent Seahorse XFp mini culture plates (8-wells) up to 70% confluency and allowed to grow at 37°C in 5% CO2 incubator, as per the manufacturer’s protocol. For the cell mitostress assay, the sensory cartridge was hydrated with milli-Q water one day before at 37°C incubator without COth day. The experiments were performed for three times, the total cell count after each day of infection was taken and an average of two experiments were considered using Microsoft Excel software.2 sets of 60mm dish , (∗∗), (∗∗∗) signifies p-values of 0.05,0.005 and 0.0001. (ns) signifies non-significant. All densitometry analysis was done using Image J software.The graphs were presented using graph pad prism 9. Experiments were performed at least 3 times and data were represented as means and standard deviations and were statistically analyzed by Student’s


However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.Zinc (Zn ZIP+ levels . Several+ levels , 36, suggranules , 18, 37.x status . The mec22.1et Nakagata, N.  were originally described by Dufner-Beattie et al.  according to the manufacturer’s instructions including a genomic DNA digest. Reverse transcription up to 1 µg of RNA was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative PCR (qPCR) was performed with SYBR Select Master Mix on a ViiA7 (Life Technologies). Gene expression was calculated by the comparative ΔΔCt-method and normalized to the housekeeping gene Rplp0. All primers had melting temperatures of 58-60°C .2.42+-free Tyrode’s buffer containing PGI2 (0.1 μg/ml) and apyrase (0.02 U/ml) and centrifuged at 2,800 rpm for 5 min at RT. Finally, the platelet pellet was resuspended in the appropriate volume of Ca2+-free Tyrode’s buffer containing apyrase (0.02 U/ml) to reach the required platelet concentration for experiments. Of note, platelet agonist concentrations might vary due to the experimental conditions.Murine blood was collected from the retroorbital plexus into 1.5 ml reaction tube containing 300 μl heparin in TBS . To prepare platelet-rich plasma (PRP), 200 μl TBS/heparin were added and blood was centrifuged at 800 rpm for 6 min at RT. Then, supernatant and buffy coat were transferred into a new tube containing 300 μl TBS/heparin and centrifuged at 800 rpm for 6 min at RT. To prepare washed platelets, PRP was centrifuged at 2800 rpm for 5 min at RT. The platelet pellet was gently resuspended in 1 ml Ca2.56 cells/µl) were resuspended into sterile 0.9% saline solution without BSA. The cell suspension was centrifuged at 2,000 rpm for 2 min to get platelet pellets which were dissolved in 13.8% ultra-pure HNO3 and left for 75 min at 90°C. The lysed cells were stored at -20°C until measurements of Zn2+, Fe2+, Ca2+ and Mg2+ ion levels with ICP-MS . Before the measurements, the lysates were diluted 1:20 into ultra-pure water to be at final concentration of 0.69% HNO3. Five standard solutions were prepared individually for each element containing 10, 1, 0.1, 0.01 and 0.001 mg/l of Zn2+/Fe2+/Ca2+ ions or 100, 10, 1, 0.1 and 0.01 mg/l Mg2+ ions . The blank containing 0.69% HNO3 was subtracted from each standard solution and sample. The internal Rh standard  with a concentration of 10 µg/l was automatically added to each sample by the ICP-MS device. The measured ion concentration in mg/l was multiplied with 20, which means that the displayed ion concentration corresponds to the concentration in the cell lysate solution in 13.8% HNO3.Washed platelets (1x102.65 cells/µl) were loaded with FluoZin-3 (2 µM) for 30 min at 37°C in the dark. Fluorophore-loaded platelets were washed, centrifuged at 2,000 rpm for 2 min and resuspended with Tyrode’s HEPES buffer. 25 µl of loaded platelets were diluted with 1 ml of Tyrode’s HEPES buffer. Since ZIP1/3 DKO mice express very low levels of GFP from the Slc39a1 and Slc39a3 gene locus (2+ after stimulation was determined as (F2-F0)/(F1-F0)*100 and the relative release as (F1-F2)/(F1-F0)*100.Washed platelets  and incubated for 30 min at 37°C. Reaction was stopped with 1.5 ml Tyrode’s without Ca2+. Measurements and analyses were performed analogously to FluoZin3 measurements, with gating on 50 μl of whole blood was washed twice  in Tyrode’s buffer without Ca2.86 platelets/μl were lysed in buffer  containing 1% Igepal and protease inhibitors, incubated for 20 minutes at 4°C, and centrifuged 10 min at 14,000 rpm. For determination of Von Willebrand factor (vWF) content, 96-well plates were coated with 10 μg/ml rabbit anti-human vWF (DAKO #A0082) overnight at 4°C and blocked with 5% BSA for 2 hours at 37°C. Sample dilutions (1:200) were incubated for 2 hours at 37°C. After washing with TBS containing 0.1% Tween, samples were incubated with HRP-coupled rabbit anti-human vWF antibody  for 2 h at 37°C. After washing, ELISAs were developed using Ultra-Sensitive TMB (tetramethylbenzidine) substrate (Europa Bioproducts) and the reaction was stopped with 0.5 M H2SO4. Adsorption was determined at 450 nm and 620 nm. Background signals (wells with no sample added) were subtracted from all samples.Washed platelets at a concentration of 1×102.92+ was added. 50 μl of diluted blood were stained for 15 min at RT with saturating amounts of fluorophore-conjugated antibodies and analysed directly after addition of 500 μl PBS. To analyse platelet activation responses, blood samples were washed twice  in Tyrode’s buffer without Ca2+ and finally resuspended in Tyrode’s buffer containing 2 mM Ca2+. Platelets were activated with appropriately diluted agonists for 8 min at 37°C followed by 8 min at RT in the presence of saturating amounts of PE-coupled JON/A  detecting activated αIIbβ3 integrin, and FITC- or DyLight649-coupled anti-P-selectin  antibodies in the dark. To study the involvement of PKC, ERK1/2 or CamKII signaling pathways, platelets were pre-incubated for 1h at RT with bisindolyl-maleimide-1 (10µM), UO126 (5µM) or KN-93 (10µM), respectively. The reaction was stopped by addition of 500 μl PBS and samples were analysed with a BD FACSCelesta. Platelets were identified by their forward/side scatter (FSC/SSC) characteristics. Obtained data was analysed using FlowJo . For FITC, background MFI of unstained platelets was subtracted.Measurement of platelet glycoprotein expression has been described recently . Briefly2.102+ at a concentration of 2x105 platelets/μl, were transferred into a cuvette. For all measurements with washed platelets, except those with thrombin as agonist, Tyrode’s buffer was supplemented with 100 μg/ml human fibrinogen. Agonists were added as 100-fold concentrates and light transmission was recorded over 10 min with an Apact 4-channel optical aggregation system . For calibration of each measurement, Tyrode’s buffer was set as 100% aggregation and washed platelet suspension was set as 0% aggregation, before the agonist was added.200 μl of washed platelets in Tyrode’s buffer containing 2 mM Ca2.112+) were stimulated with thrombin (0.01 U/ml) and immediately added to the fibrinogen surface. Platelets were allowed to adhere and spread at 37°C for 10 min. The non-adhered platelets were washed away and the adhered cells were fixed by addition of 300 μl 4% PFA/PBS for 10 min at RT. Then, the wells were washed twice with 300 µl PBS. Platelets were visualized by differential interference contrast (DIC) microscopy with a Zeiss Axiovert 200 inverted microscope (100x/1.4 oil objective). Representative images were taken using a CoolSNAP-EZ camera  and evaluated according to different platelet spreading stages with ImageJ (NIH)  . Spreadi2.125/μl in Ca2+-free Tyrode’s buffer. 100 μl of this suspension were loaded with 3 μM fura-2-AM in the presence of 0.2 μg/mL pluronic F-127  for 20 min at 37°C. After labelling, the platelets were washed once and resuspended in 500 μl Tyrode’s buffer containing 1 mM Ca2+ for measurement of Ca2+ influx. This cell suspension was transferred into a cuvette and fluorescence was measured with a PerkinElmer LS 55 fluorimeter  under stirring conditions. Excitation was switched between 340 and 380 nm and emission was measured at 509 nm. Basal Ca2+ levels were recorded for 50 s before the indicated agonist was added. Each measurement was calibrated using 1% Triton X-100 and EGTA according to (Washed platelets were adjusted to a concentration of 2.5 x 10rding to .2.135/µl in BSA- and Ca2+-free Tyrode’s buffer and supplemented with apyrase (2 mM f.c.). Platelets were stimulated under stirring conditions in an aggregometer at 37°C, with different agonists, as indicated in the figures. The stimulation was stopped by mixing the platelet suspension with 10x concentrated ice-cold cell lysis buffer  containing Pefabloc SC-Protease-Inhibitor and phosphatase inhibitors (Carl Roth) and kept on ice for at least 30 min. Afterwards, platelet lysates were mixed with 4x SDS sample buffer (1x f.c.), boiled for 5 minutes and subjected to Western blot analysis. Samples were separated by SDS-PAGE using a 10% separating gel for electrophoresis, followed by transfer onto a PVDF membrane. Membranes were stained with Ponceau S to prove equal loading, blocked for 10 minutes in 10% BSA in TBS-T and then incubated overnight at 4°C with the indicated antibodies obtained from Cell Signaling Technology (phospho-PKC substrate motif (#6967), phospho-CamKII (#12716), phospho-p42/44 MAPK (#4370), β-actin (#3700)) or Merck/Sigma-Aldrich (tubulin (#T6074)). Afterwards, the membranes were washed three times with TBS-T at RT before being incubated with the appropriate secondary HRP-labelled antibodies (CST #7074 or #7076) for 1 h at RT. Finally, the membranes were washed several times and proteins were visualized using the Clarity™ or Clarity™ Max Western ECL Substrate (Bio-Rad) and the ChemiDoc Imaging System (Bio-Rad). For relative quantification, densitometric analyses of Western blots (non-saturated chemiluminescence and Ponceau S stainings) were carried out using ImageJ   for 5 min at 37°C. The diluted blood was filled into a 1 ml syringe and connected to a transparent flow chamber with a slit depth of 50 μm, equipped with the coated coverslips. Perfusion was performed using a pulse-free pump under high shear stress equivalent to a wall shear rate of 1,000 s-1 for 4 min. Thereafter, coverslips were washed by a 4 min perfusion with Tyrode’s buffer at the same shear rate and phase-contrast and fluorescent images were recorded from at least five different fields of view (63x objective) using a Leica DMI6000 microscope. Image analysis was performed using ImageJ (NIH) (Coverslips (24x60 mm) were coated with 200 μg/ml fibrillar type-I collagen (Horm) overnight at 37°C and blocked for 1 h with 1% BSA at RT. Blood (700 μl) was collected into 300 μl heparin in TBS  and two parts of blood were diluted with one part Tyrode’s buffer with 2 mM CaeJ (NIH) , 44. Thr2.152 filter paper saturated with 20% FeCl3. Adhesion and aggregation of fluorescently labelled platelets  in arterioles was monitored for 40 min or until occlusion occurred (blood flow stopped for >1 min). Digital images were recorded and analysed using MetaMorph software (Molecular Devices).Experiments were performed as described recently . Briefly2.16The shown data are expressed as mean ± SD or ± SEM, where appropriate. When applicable, a modified Student’s t-test was used to analyse differences between two groups. For analysis of more than two groups, two-way ANOVA for repeated measures followed by the Bonferroni multiple comparison post-hoc test was applied, p-values <0.05 were considered as statistically significant (*), p<0.01 (**) and p<0.001 (***).33.12+ in the regulation of platelet activity and signalling  family of transporters, which regulate the import of Zn2+ into the cytosol  or ZIP1/3 DKO Slc39a1  as critical for platelet dense granules biogenesis . To asse2+ influx into the cytosol, we determined total levels of intracellular Zn2+ (protein-bound and labile) by inductively coupled plasma mass spectrometry (ICP-MS). No significant differences were found for overall [Zn2+]i or other divalent cations  , ADP (0.5 µM) and the thromboxane A2 (TxA2) analogue U46619 (0.2 µM)  in flow cytometry using the JON-A/PE antibody, which binds αIIbβ3 only in its active conformation (IIbβ3 integrin activation in response to threshold concentrations of thrombin (0.002 U/ml)  in platelets from ZIP1/3 DKO and WT mice     , we asse3.42+ is considered to be an important contributor to haemostasis and thrombosis  transcription during in vitro differentiation of MKs from haematopoietic stem cells (HSCs) (data not shown). Similarly, we did not observe any defects in other blood cells which is in line with the initial description of the ZIP1/3 DKO mouse line , Slc39a6 (ZIP6), Slc39a7 (ZIP7), Slc39a9 (ZIP9), Slc39a10 (ZIP10) and Slc30a1 (ZnT1) mRNA in MKs . Since FluoZin3-stainable free Zn2+ appears to be largely sequestered in granules  was calculated to result in 1300 nM [Ca2+]i, which makes a significant unspecific detection of Zn2+ by Fura-2 in this context very unlikely.So far, no data were available about the mechanisms by which Znlatelets , 11, 14,latelets , or mighlatelets . In addi cations –60. Inte2+ and Ca2+ pathways converge is PKC. It has been reported that Zn2+ augments PKC activity in lymphocytes and platelets  in heterologous cell systems by promoting the interaction of STIM1 and Orai1 in a positive feedback loop  and MEK1/2 inhibitors attenuated agonist-induced cPLA2 phosphorylation and downstream thereof TxA2 secretion in stimulated platelets , a hallmark of immunothrombosis (2+ overload and Zn2+ deficiency modulate NET formation (2+ concentrations by release from platelets might modulate this process. One consequence of NET formation is the activation of FXII, which can trigger the contact activation pathway and the kinin-kallikrein pathway and is suspected to contribute to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19 (2+ is required for autoactivation of FXII, and because platelets are able to modulate FXII function in a Zn2+-dependent manner (2+ release and alterations in the microenvironment impact FXII activation in future studies.Besides these platelet intrinsic contributions of Znrombosis , 82. Altormation , implyinCOVID-19 . Since Zt manner , 85, it In vivo, ZIP1/3 DKO mice develop faster thrombi in response to vascular injury. In vitro data show that platelets from ZIP1/3-deficient mice are hyperreactive to threshold concentrations of GPCR agonists, in particular thrombin, while responses to ITAM-coupled receptor agonists appear to be unaffected. Thereby our data encourage future studies to delineate the possible exclusive involvement of individual Zn2+ transporter in platelet activation in response to different agonists and their role in thrombosis. This holds particularly true for situations that require a balanced contribution of platelets to the immune response as in immunothrombosis or thrombo-inflammation.Taken together, our study for the first time attributes an important regulatory role to zinc transporters in platelet activation. The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Regierung von Unterfranken.AE, PÖ, ME, KM, FK, CK, UG, TV and HH performed experiments and analysed data. HS analysed data and together with BN discussed results, provided scientific input and technical equipment throughout the study. MRB generated the mice. AE, TV, HH wrote the manuscript with input from all the authors. AE, TV and HH conceived the study and designed research. All authors contributed to the article and approved the submitted version.


Although about 100 binary borides have been explored, only a few of them show superconductivity with relatively low Tc. In this work, we report the discovery of superconductivity up to 32 K, which is the highest Tc in transition-metal borides, in MoB2 under pressure. The Tc of MoB2 in the α phase can be well explained by theoretical calculations in the framework of electron-phonon coupling. Furthermore, the coupling between the d electrons of Mo and the out-of-plane Mo-phonon modes are the main driving force of the 32 K superconductivity of MoB2. Our study sheds light on the exploration of high-Tc superconductors in transition metal borides.Since the discovery of superconductivity in MgB 2 under pressure and highlight roles of transitional metals in the high-Tc superconductors.Discover the superconductivity up to 32 K in MoB Tc) are long-sought targets in the condensed matter physics and materials science communities. Materials with light elements [Tc according to Bardeen-Cooper-Schrieffer (BCS) theory [2 at 39 K in 2001 has reinforced the scientific importance of metal diborides (MB2) [MB2 show superconducting behavior. Furthermore, the Tc of these superconducting MB2 compounds are much lower than that of MgB2 (Superconductors with high transition temperature (high elements  are espe) theory . In vari of MgB2 .2) is unique among the MB2 family since it is the only material that has two structural forms: the α-MoB2 phase  [2 phase  graphitic boron layers. In contrast, there are two different kinds of B layers in β-MoB2   and β-Mocument}) . AlthougoB2 Fig. : one forers Fig. . MoB2 ma2 under various pressures. We find that MoB2 exhibits superconductivity under high pressure, reaching a Tc of 32 K around 100 GPa, which is the second highest Tc among all known boride superconductors. Synchrotron X-ray diffraction (XRD) measurements indicate that β-MoB2 transforms to α-MoB2 around 65 GPa. Although the compressed MoB2 has the same structure and comparable Tc with MgB2, the superconducting mechanism of the former is completely different from that of the latter. Theoretical calculation suggests that the d electrons and phonon modes of transition metal Mo atoms play utterly different roles in the emergence of superconductivity in contrast to the dominance of p electrons and phonon modes of B atoms in the superconductivity of MgB2.Pressure, as a conventional thermodynamic parameter, is a clean and powerful tool to tune the electronic properties of materials. It is possible to trigger structural and electronic transitions, subsequently inducing novel quantum phenomena . Here wein situ XRD measurements on the structural evolution of MoB2 under various pressures. As shown in Fig. 2  of MoB2 with lower enthalpies than the 2 structure  of a β-MoB2 single crystal at various pressures. Figure T) curves for pressure up to 109.7 GPa. The ρ(T) curves display a metallic-like behavior in the whole pressure range. When the pressure increases to 21.7 GPa, a small drop of ρ is observed at the lowest measuring temperature (Tmin = 1.8 K), as shown in Fig. P > 38.5 GPa, indicating the emergence of superconductivity. The superconducting Tc increases dramatically with pressure. Beyond the critical pressure (Pc = 70 GPa), where the structural phase transition happens, the growth of Tc slows down and the maximum Tc of 32.4 K is attained at P = 109.7 GPa, which is the highest pressure we can exert on the sample. At this pressure, the superconducting transition width ΔTc [Tc. Figure T) should originate from a superconducting transition. The derived upper critical field μ0Hc2(T) as a function of temperature T can be fitted well using the empirical Ginzburg-Landau formula  = μ0Hc2(0)(1 − t2)/(1 + t2), where t = T/Tc is the reduced temperature with zero-field superconducting Tc. The fitted zero-temperature upper critical field μ0Hc2(0) of MoB2 from the 90% ρn criterion can reach 9.4(1) T with Tc = 31.7(2) K, which is consistent with the measured value of 32.4 K.Since MoB2 K Fig. , which iula Fig.  μ0Hc2(T)2 for five independent runs provide consistent and reproducible results  the growth of Tc slows down (0.1 K/GPa). The Tc of MoB2 rises to as high as ∼32 K at a pressure of 109.7 GPa and still does not exhibit the trend of saturation.The measurements on different samples of MoB results , confirmTc superconductivity in MoB2 under pressure, we performed density functional theory (DFT) [2 in the α phase at 90 GPa, for which the experimental Tc can reach ∼31.5 K at 91.4 GPa. The calculated band structure of α-MoB2 at 90 GPa along the high-symmetry paths of the Brillouin zone is shown in Fig. EF), indicating its metallic character. Based on the analysis of the total and partial density of states  have larger contributions than B p orbitals around EF. The phonon dispersion and the phonon density of states F(ω) for α-MoB2 at 90 GPa are displayed in Figs. 2F(ω)  ,16 and dry (DFT) ,18 calcutes Fig. , Mo d or formula ,20, we c to 0.15 ,20, we o.3 K see , showing2 at high pressure shares the same crystal structure as MgB2 and owns a comparable superconducting Tc with that of MgB2 at ambient pressure [2, the p orbitals of B atoms in the B-B honeycomb lattice contribute most around the Fermi level [2 under high pressure, the states near EF are dominated by the d electrons of Mo atoms  at the Fermi level  could increase along with more planar boron layers  mentioned above probably have a close connection with the increase of Tc in β-MoB2 in the phonon-mediated superconductivity regime.In addition to the α phase of MoBsure see , which asure see . The smami level , which on layers , hence ffunction , as showure Fig. , suggest2 under pressure. Although compressed α-MoB2 and MgB2 both possess the AlB2-type structures and comparable Tc, the features of their electron-phonon couplings that mediate the superconductivity are quite different. In both MgB2 and α-MoB2, the active phonon modes directly modulate the active electronic bands, causing the strong electron-phonon coupling [d electrons of Mo in α-MoB2, in comparison with the stretching B-phonon mode modulating the p orbitals of B in MgB2. Thus, not only do the electrons in light-element atom networks need to be considered, but the relatively localized electrons of metal atoms may also be worthy of attention in the search for high-Tc superconductors.In summary, we found superconductivity up to 32 K in MoBcoupling . However2 were grown by Al flux (see the In situ high-pressure XRD and resistivity measurements were performed in a diamond anvil cell. We employed the swarm-intelligence-based CALYPSO structure prediction method [2 under high pressure. The electronic structure, phonon spectrum and EPC of AlB2-type α-MoB2 at 90 GPa were studied based on DFT [High-quality single crystals of MoB see the . In situn method  to find d on DFT ,16 and Dd on DFT ,18 calcu2 by another group [2 via the application of external pressure [After we submitted this paper, we learned that pressure-induced superconductivity was observed in WBer group . Most ofer group . We alsopressure .nwad034_Supplemental_FileClick here for additional data file.


In this study, we test the effect of the stoichiometric ratio of ethylene carbonate (EC) as an organic additive on the electrochemical performance of halogen-free electrolyte (HFE) based on Mg(NO3)2 in acetonitrile (ACN) and tetraethylene glycol dimethyl ether (G4). Through various characterization methods, the introduction of EC perturbs the bonding scheme of the HFE electrolyte, enhances the ionic conductivity, reduces the relaxation time, and forms a resistive solid electrolyte interphase (SEI). The assembled Mg–S full cell using modified HFE (HFE_EC) delivers initial specific capacities of 900 m Ag−1 with a cycle life of up to 10 cycles in the case of activating the cell with electrochemical conditioning. This study sheds light on the interplay of EC and the interfacial kinetics in Mg batteries and opens a door for designing novel magnesium electrolytes.Magnesium metal batteries attract great attention for their high volumetric capacity and safety as a post-lithium choice. The strategy of adding organic plasticizer may bring new insights into designing halogen-free electrolytes for the further development of magnesium–sulfur batteries. The high charge density of Mg Ethylene carbonate perturb the bonding scheme of HFE and catalyze the dissociation of the dissolved salt and hence electrolyte conductivity. However, there are technical challenges like the poor cyclability, the shuttle effect of polysulfides, and the highly resistive passive anode/electrolyte interface that must be overcome before the practical transition from the lab to the market.1 BaTiO3 possesses a piezoelectric property, generating electrostatic charges on the surface under electrochemical stress. This property gives BaTiO3 the ability to immobilize the polar polysulfide molecules via chemical adsorption, reducing polysulfide shuttling and improving the cycle life of Mg–S cells.2,3,4 The electrolyte controls the electrochemical activity, safety stability, and thermodynamics of the batteries. Therefore, designing an efficient magnesium electrolyte compatible with sulfur's electrophilic nature will be essential to achieving a practical Mg–S battery. In this regard, non-nucleophilic electrolytes were designed, including hexamethyldisilazide-Br,5 non-nucleophilic Mg(TFSI)2–MgCl2–1,2-dimethoxyethane/tetrahydrofuran,6 magnesium tetrakis(hexafluoroisopropyloxy)borate,7 magnesium perfluorinated tert-butoxide (Mg(pftb)2),8 Mg[B(Ohfip)4]2.9 Nevertheless, the common factors of the most weakly coordinating Mg salts are the low stability, high cost, and complex synthesis process, that impede their practical applications. In our previous research,10 we reported a simple halogen-free electrolyte (HFE) 0.69 M Mg(NO3)2·6H2O in ACN : G4 (∼2 : 1) with ionic conductivity of ∼10−4 S cm−1 at 313 K, dendrite-free, and translucent Mg deposits. However, much work need to be dedicated to lowering the Mg2+ desolvation energy and reducing the Mg stripping/plating overpotential. Ethylene carbonate (EC) organic cosolvent is commonly used to tailor the interfacial kinetics and the transport properties of the electrolyte and played a vital role in the commercialization of lithium-ion batteries. The high dielectric constant of EC grants it the ability to dissociate metal salt and engineer the solid anode/electrolyte interphase.11,12 Herein, we study for the first time the beneficial effects of the interplay between HFE and EC additive and how it affects the Mg2+ desolvation energy. The study was revealed by UV-vis spectroscopy, Fourier transforms infrared (ATR-FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). Galvanostatic cycling was conducted on the Mg/Mg symmetric battery with the HFE_EC electrolyte to test the change of the overpotential with introduction of EC. The nature of Mg/HFE_EC interface was probed using FTIR, X-ray diffraction (XRD), and SEM_EDS techniques. Mg–S cells with HFE and HFE_EC electrolytes were assembled and tested electrochemically, and the sulfur cathode was retrieved from the disassembled cells to examine the morphology and structure of the S cathodes at different electrochemical states using SEM, EDX, XRD and EIS. The study confirms the impact of EC additive to regulate the electrochemical performance of the HFE via modulating Mg2+ desolvation barrier but it results in undesirable SEI which causes high Mg stripping/plating overpotential.The transition from fossil fuel to green fuel depends on the ability to bridge the gap of renewable energy intermittency through sustainable energy storage systems. Mg–S batteries are an attractive option due to their low cost, environmental sustainability, and abundant magnesium and sulfur in the Earth's crust. In addition, Mg–S batteries offer competitive theoretical energy density (1684 W h kg3)2·6H2O dissolved in ACN : G4 (∼2 : 1) was synthesized according to the published work.10 Five bottles of HFE were prepared and x M of EC,  was added to each one, which were denoted as HFE, HFE_X1, HFE_X2, HFE_X3, and HFE_X4 respectively. ATR-FTIR measurements were recorded in the wavenumber ranging from 400 to 4000 cm −1 using ALPHA II Bruker spectrometer. UV-vis spectral data were collected using Edinburgh DS5 Dual Beam UV-vis spectrophotometer. The S cathode was prepared by grinding 75 wt% of sulfur  with 20 wt% graphene nanoplatelets , and 5 wt% BTO (Alfa Aesar 99%). The resultant mixture was placed in the microwave for 10 seconds and then subjected to a ball mill for 30 hours to obtain the S_GNP_BTO active cathodic material. 0.75 g S_GNP_BTO : 0.1 g Super CP : 0.15 g polyvinylidene fluoride was dissolved in N-methyl-2-pyrrolidinone using a magnetic stirrer. The resultant viscous slurry was spread on Al foil with a thickness of 100 μm using HOHSEN MC-20 Mini-Coater and left in the oven for two hours at 100 °C. After that, the cathode was cut with a diameter of 14 mm and then permanently kept at a temperature of 65 °C. Electrochemical impedance spectroscopy (EIS) and electrochemical measurements of the electrolyte were tested using CHI604E electrochemical workstation. The collected impedance data were analyzed by ZView software. The galvanostatic test was conducted between 0.25 and 2.5 V using a NEWARE BTS4000. XRD pattern was recorded on Rigaku MiniFlex 600 diffractometer, while SEM images were captured using Jeol JMS-700 EDS electron microscope.The halogen-free electrolyte (HFE) based on 0.69 M Mg scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching mode of pure EC.14 Furthermore, the introduction of the EC in the structural skeleton of the HFE results in additional small new peak characteristic of –C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N⋯Mg2+ at wavenumber 2362 cm−1, which indicates the role of EC in engineering electrolyte solvation structure via supporting weak solvent–cation interaction, promoting the rapid desolvation of Mg2+ ions.15 The UV-visible absorption spectra are a powerful tool to analyze the band structure of the energy materials. xEC electrolytes in the 200–1100 nm range. The band gap energy Eg was estimated using Tauc's relation:16αhν = β[hν − Eg]n, hν versus (αhν)0.5 for HFE_xEC electrolytes. The Eg estimated from the extrapolation of the linear region of the plot on the x-axis (αhν = 0) gives the value of the optical bandgap, Eg. The bandgap was found to be around 3.43 eV of the HFE_xEC electrolytes; hence HFE_xEC shows the difficulty in losing electrons (non-nucleophilic), which makes it a compatible electrolyte with the electrophilic nature of the S8. versus the wavelength (λ) of HFE_xEC electrolytes. The value of k shows a high dispersion with increasing wavelength in the UV period (200–340 nm) and is followed by a little increase in the visible region. Also, it can be noticed that the value of k relatively increases with increasing EC content. The refractive index (n) is an optical property that measures the change in the velocity of light inside the electrolyte, which can be calculated using the equation:17,18, where R = [1−(A + T)], A and T are the absorption and transmission coefficient, respectively. versus the wavelength (λ) of the relation between the refractive index (n) of HFE_xEC electrolytes. The profile shows increasing the ability of the HFE_xEC mediums to bend the path of the electromagnetic waves with the transition from UV to the visible regions and increasing EC content. This indicates EC's role in changing the optical density and electronic structures of HFE. Rb (due to migration of ions) and bulk capacitance (due to immobile species), whereas the low-frequency spike is due to the interfacial polarization effect.19 The bulk conductivity was calculated by fitting the semicircle curves using ZView software and substituting in the following eqn: , where L and A are the thickness and cross-sectional area of separator, respectively. Arrhenius equation:  was used to calculate the activation energies Ea of ion conduction,  at 100 Hz of HFE_xEC electrolytes, where σo, and K represent the pre-exponential conductivity factor, and the Boltzmann constant. The calculated activation energies are 0.052, 0.076, 0.075, 0.07 and 0.067 eV for HFE, HFE_X1, HFE_X2, HFE_X3, and HFE_X4 respectively. The behavior of the activation energy with the increasing the content of EC can be understood in the light of the low concentration of EC was not enough to dissociate the ions.The functional group's evolution and possible molecular interaction of HFE_ε′ versus imaginary dielectric loss ε′′ of HFE_xEC electrolytes at 303 K, and each plot appears as a semicircle and intersects the ε′-axis. Relaxation time τ was calculated using the relation τωmax = 1, where ωmax is the angular frequency of the maximum ε′′. 4/Mg cells. The values of the Mg2+ transference number t2+Mg was calculated using Bruce–Vincent–Evans equation: , where Is and I0 are steady and initial state current, respectively, and ΔV is the applied voltage (0.01 V). The Mg-ion transference t2+Mg values were recorded to be ∼0.77 and 0.866 for HFE, HFE_X4 electrolytes, respectively. The existence of the slope in the polarization curves is based on the reduction of the electrolyte with the high charge density electrode . Future work is in progress to protect the surface of the Mg anode from direct contact with the electrolyte solution or use functional additives to inhibit this reduction. The oxidative stability (−1–4.5 V (vs. Mg/Mg2+)) of the HFE and HFE_X4 electrolytes were measured by linear sweep voltammetry (LSV) in stainless-steel/HFE/Mg and stainless-steel/HFE_X4/Mg cells, 4/Mg cells with different rest time at open circuit voltage. The diameter of the semicircle decreases with an increase in the rest time, then decreases with a further increasing the rest time, and eventually stays at a stable value implying a stable interface between the electrolyte and the Mg-metal electrode. 4/Mg cells before and after cycling. The plots were fitted using ZView software with the equivalent circuit shown as inset RSEI) of 34/78 Ω and a charge transfer resistance (Rct) of 10 089/8949 Ω in the pristine state. However, the cell with modified electrolyte (HFE_X4) possessed clear semicircles before discharge and open semicircle after discharge and significantly lower resistances before discharge and higher resistance after discharge compared with the cell with the pristine electrolyte which confirms that the modified electrolyte decomposed to a resistive SEI.20,214 at 0.2 mA cm−2. Mg/HFE/Mg shows a stable voltage profile with low overpotential compared with symmetric Mg/HFE_X4/Mg cells, which show higher overpotential overgrowing with cycling. The results confirm that the EC reacts with the Mg anode during cycling, producing resistive byproducts that might have negatively affected the cycle life.224/Mg symmetric cells after cycling. The surface of pristine Mg shows flat morphology, while that of Mg_HFE displays low-density stick morphology on the front-based Mg and the residual Si from the fiberglass separator. The surface of Mg_HFE_X4 shows high-density thick stick morphology compared with that of Mg/HFE. EDS analysis of bare Mg, Mg_HFE, and Mg/HFE_X4, as shown in 4. The results confirm that the nature of the anode/electrolyte interface differs in the presence of EC, which reacts with Mg anode during cycling producing a passive byproduct as mentioned before. 4/Mg symmetric cells after cycling. The reflection patterns in the three samples are the same and are centered on 2θ = 34.3°, 32.2°, 36.6°, 47.8°, and 63° assigned to the crystalline planes of (002), (100), (101), (102) and (103), respectively,  of the hexagonal Mg structure (JCPDS No. 901-3058). To assess the electrochemical performance of the optimized electrolyte with the designed sulfur cathode, CV tests of Mg/HFE/S and Mg/HFE_HFE_X4/S cells were conducted in the potential range of 0–2.6 V at a scan rate of 8 mV s−1, 4 has an observable anodic peak at ∼2 V and a higher cathodic/anodic current compared with the cell with HFE. However, this behavior confirms that electrolytes decomposed to an unstable SEI. EIS is regarded as one of the most powerful tools to analyze the cathode kinetic performance. The Nyquist plots of Mg/HFE/S and Mg/HFE_X4/S cells before and after short cycling in the frequency range between 1 Hz and 1 MHz are shown in Z′ at high frequency, semicircle at high-range frequency, semicircle at middle-range frequency, and a sloping line at low frequencies assigned to the bulk resistance of the electrolyte, diffusion, and migration of Mg2+ through the solid electrolyte interphase on the surface of the electrode (RSEI), charge transfer resistance (Rct) and to the Warburg impedance (represents magnesium-ion diffusion through the sulfur electrode), respectively. EIS parameters were calculated via fitting the equivalent circuit (Randell circuit model) using ZView software and the obtained parameters are displayed in 2+ (DMg2+) was calculated by eqn:23, from linear fitting the of the relation Z′ = Rs + Rct + σω−0.5 (ω−0.5vs. Z′), the Warburg factor σ was calculated from the slope at low frequencies, D2+Mg for all the cells are around 6 × 10−15 cm2 s−1. −2 for Mg/HFE/S and Mg/HFE_X4/S cells. The EC-containing electrolyte delivered a high discharge/charge capacity of ∼3000/1450 mA h g−1 over the theoretical capacity of the S electrode (∼1673 mA h g−1), while the pristine electrolyte (HFE) delivered reasonable discharge/charge below the theoretical capacity of sulfur with short cycle life. Thus, we can conclude that the two cells' major discharge/charge capacity value is due to the electrolyte decomposition. Aiming to improve the cycle life of the developed electrolyte in the Mg–S cells, galvanostatic cycling was considered as a conditioning process to scavenge the active contaminants and partially remove the passivation from the surface of the anode. vs. specific capacity with a current density of 0.02 mA cm−2 of Mg/HFE_X4/S cell after pre-activating using the electrochemical conditioning processes. The activated cell delivered an initial discharge/charge capacity of ∼875/355 mA h g−1, and the cycle life extended to 10 cycles with coulombic efficiency >100%, suggesting that the electrolyte decomposition is still the major factor that controls the cycle life even with the electrochemical conditioning processes. The XRD was conducted to probe the crystal structure changes of the sulfur cathode from pristine → discharge → recharge states, 0 (pristine cathode) shows the main peak locations at 23°, 25.74°, 28.58°, which matches the 222, 026, and 313 reflection signals of S8 with orthorhombic S lattice , confirming that sulfur is the main constituent of the cathode.24 After discharge, all peaks showed intense broadening indicating the formation of an amorphous electrolyte/cathode interface and the reaction of sulfur atoms with Mg2+. θ = 23° in the pristine state. The peak shifted to a low angle after discharge while it showed further reduction and a relatively a small shift to the original position after recharge indicates a partially reversible conversion reaction. The Williamson–Hall (W–H) model was applied to estimate the uniform stress deformation and the S cathode's average crystallite size (D) at different electrochemical states. The formula for W–H model is expressed as:  where β is the full width at half maximum (FWHM), θ is the Bragg's angle, k is the shape factor (0.94), and λ is the X-ray wavelength. β cos(θ) versus 4 sin(θ) of S0, S1, and S2, the slope of the line and the y-intercept represent the strain and crystallite size, respectively.25 The lattice strain values and crystallite size are listed in 2+ within the sulfur skeleton. SEM images and EDS results of the S0, S1, and S2 composite are shown in 0 electrode shows an inhomogeneous distribution of sulfur deposits on the GNPs network surface. After magnesiation (S1), it can be noticed that the Mg2+ ions interact with S atoms and show stick sediment distribution. The stick sediments disappeared in de-magnesiation state (S2), and the surface showed smooth morphology. 0, S1, and S2, respectively. By following the evolution of Mg2+ ratio that increased from zero in the pristine state to 16.32 wt% upon discharge and decreased to 11.48 wt% upon recharge state, the results support the partially reversible conversion reaction of the S8 to polysulfide molecules and vice versa. Furthermore, the evolution of the sulfur ratio from 18 wt% in S2 after discharging to 6 wt% upon subsequent charge process confirms the shuttling of polysulfide, which is presumably responsible for the capacity fading.3)2 in acetonitrile (ACN) and tetraethylene glycol dimethyl ether (G4). The FT-IR spectra confirm that EC additive changed the Mg2+ solvation structure via supporting weak solvent–cation interaction, promoting the rapid desolvation of Mg2+ ions. Furthermore, the addition of EC enhanced the ionic conductivity and Mg2+ ion transference number of the electrolyte, which can be understood from the role of EC in disassociating the magnesium salt and forming SEI. However, the introduction of EC results in the formation of a resistive SEI, causing high Mg stripping/plating overpotential. The Mg–S full cell using modified HFE (HFE_EC) delivers an initial specific capacity of 900 mA g−1 with a cycle life of up to 10 cycles in case of activating the cell with electrochemical conditioning. Much work must be dedicated to optimizing the EC-based halogen-free electrolytes to realize practical Mg–S batteries.In this work, we investigated the efficiency and electrochemical compatibility of EC additive to halogen-free electrolyte based Mg(NOThere are no conflicts to declare.