While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.KCNQ family K 2 structures and identify CP1 as a compound that could restore KCNQ1 currents after PIP2 depletion. Like PIP2, this compound can mediate VSD-pore coupling of the channel, albeit via a different mechanism from PIP2.Yongfeng Liu, Xianjin Xu, Junyuan Gao et al. perform in silico screening targeting compounds that resemble both KCNQ1 and PIP V7.1-5) are important in regulating cardiac action potential duration3, modulating neuroexcitability6, and maintaining endolymph K+ homeostasis in the inner ear8. The KCNQ channels are activated by membrane depolarization, but they all share an important feature in that their activation also requires the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2)12. KCNQ1 and its regulatory subunit KCNE1 form the IKs channel in cardiac myocytes that terminates action potentials and regulates heart rhythm13. Congenital mutations in KCNQ1 that alter IKs channel function are associated with cardiac arrhythmias15. Patients with mutations associated with PIP2 regulation of KCNQ1 may exhibit a high risk for life-threatening events18. The heterotetramer K+ channel formed by KCNQ2 and KCNQ3 was identified as carrying neuronal M currents4. Homotetramer KCNQ2 and KCNQ5 channels were suggested to contribute to M currents as well20. In neurons, muscarinic stimulation of Gq protein-signaling pathways hydrolyzes PIP2. The reduction of membrane content of PIP2 results in the decrease of M currents and the enhancement of neuronal excitability21. Congenital mutations that reduce M currents are associated with epilepsy and deafness12.Voltage-gated KCNQ potassium channels , and the pore is formed by S5–S6 from all four subunits. In the channel structure, four VSDs are located at the periphery of the central pore22. In voltage-gated K+ channels, membrane depolarization activates the VSDs by moving the S4 segment to the extracellular side. The conformational changes in the VSD are then coupled to the pore for channel opening. In KCNQ1 channels, numerous experiments detected that the VSD activates in two steps, first to an intermediate state and then to the activated state27. In both states, VSD activation opens the pore, resulting in the intermediate open (IO) and activated open (AO) states28. The VSD activation opens the pore via two distinctive sets of interactions. The first interactions are between the S4–S5 linker (S4–S5L) and the cytosolic end of S6 within the same subunits, and the second interactions are between the neighboring subunits involving S4 and S4–S5L in one subunit and S5 and S6 in the other27. Ion channels interact with their lipid environments during channel function. Specific lipid-channel interactions in voltage-gated K+ (KV) channels have been reported to alter VSD activation or pore opening32. On the other hand, in KCNQ1 channels, PIP2 was found to be required for VSD-pore coupling. When PIP2 is depleted from the membrane, the pore cannot open, even though the VSD remains activated by membrane depolarization18. A site at the interface between the VSD and pore was identified for PIP2 to interact and mediate VSD-pore coupling36.PIP2 mediates VSD-pore coupling in KCNQ1 remains to be understood. The structural determinants of the channel that are involved in this mechanism are not known. Further, the structural feature of the PIP2 molecule that is important for its activity is also not clear. In this study, we identified a small molecule, CP1, by screening in silico using compounds resembling the PIP2 head group to dock onto the structure model of KCNQ1, targeting the previously identified PIP2-binding site18. We found that CP1 could substitute for PIP2 to recover KCNQ1 currents that were abolished by depletion of membrane PIP2. Similar to PIP2, CP1 activated KCNQ1 channels by enhancing VSD-pore coupling. The compound also activated KCNQ2 and KCNQ3, but to an extent less than that seen for KCNQ1. On the other hand, CP1 showed significant differences from PIP2 in its characteristics and mechanism of activating the KCNQ channels. These results provide insights into PIP2 and CP1 activation of KCNQ channels, and a basis for future development of antiarrhythmic drugs that target KCNQ1 channels.The mechanism by which PIP2 for activation37. Our previous studies show that PIP2 mediates the coupling between voltage-induced VSD movements and pore opening in KCNQ1, and it may associate with the channel protein at the interface between the VSD and the pore for this function18. To further understand the effects of PIP2 on KCNQ channel activation and how the VSD-pore coupling is influenced by PIP2, we investigated if other compounds resembling PIP2 can mediate the interactions between the VSD and the pore for channel activation. We screened, in silico, a subset of the Available Chemical Database , in which each compound has a formal charge of 2 (~104 compounds), targeting the PIP2 site using both the structure-based and ligand-based methods. In the structure-based screening, compounds in the ACD subset were ranked by their binding scores calculated using an in-house docking software MDock40. In the ligand-based screening, compounds were ranked by their similarity scores with the head group of PIP2, calculated using a 3D molecular similarity program ShaEP41. The details are described in the “Methods” section.KCNQ channels require PIP2   of PIP2 and a sulfate group (S1) of CP1 bind to a similar location on KCNQ1 in distinct ways. Specifically, P1 of PIP2 forms a hydrogen bond with residue T247, but S1 of CP1 forms a salt bridge with residue R249. Last, PIP2 contains a third negatively charged group (phosphate group P5) forming a salt bridge with residue R259.From the primary screen, a compound, CP1, was found in the top 2% for both structure-based and ligand-based strategies, which contains two sulfates that may mimic the head group of PIP2 Fig. . Docking6C) Fig. . The int2-binding site reduced KCNQ1 currents18. CP1 modifies voltage-dependent activation of KCNQ1 channels by shifting the voltage dependence of channel opening, measured as the voltage dependence of the conductance (G–V) relationship, to more negative voltages  currents decrease upon co-expression with CiVSP with consecutive voltage pulses , but decreasing the current at positive voltages  shifted to more negative voltages  was 8.73 ± 0.68 µM. CP1 does not alter the ion selectivity of KCNQ1 channels  of Alexa 488 C5 maleimide attached to the S3–S4 linker, at residue C219, of the pseudo wild-type KCNQ1 (with mutations C214A/G219C/C331A) reported VSD movements in response to voltage changes, while the ionic current reported pore opening45  to measure the effects of CP1 on voltage-sensor activation. The emission  and an activated state (A state) upon membrane depolarization, and the pore can open when VSD is at either the intermediate (IO state) or activated (AO state)ion Fig. . The app mV Fig. . If the  mV Fig. , indication Fig. . In highion Fig. . The G–VCP1 Fig. . CurrentCP1 Fig. , suggestsed Fig. . Since t4. Similar to KCNQ1, these KCNQ subunits require PIP2 for voltage-dependent activation49. KCNQ2 alone can express functional channels in Xenopus oocytes, while KCNQ3 alone cannot. However, a single mutation, A315T, allows KCNQ3 to functionally express in Xenopus oocytes50. We examined the effects of CP1 on KCNQ2, KCNQ3 with mutation A315T (denoted as KCNQ3*), KCNQ2+KCNQ3, and KCNQ2+KCNQ3* expressed in Xenopus oocytes  amplitude  was not changed. However, if we continued to apply CP1 alone, the APD was significantly shortened. This indicated that CP1 effects are mediated in large part by enhancing IKs channels  to prolong action potentials and then applied various concentrations of CP1. We found that 0.2 µM CP1 was sufficient to return the action potential duration back to normal  along with the residues that interact with PIP2 and CP1  than KCNQ2 or KCNQ463. Consistently, the sites for PIP2 and CP1 interaction are generally conserved but show some differences P3, IP2, or other small phosphates had no effect on channel activity. On the other hand, any phosphate head group of a lipid could activate the channel, with PIP2 being the most effective69. Taken together with our results with CP1, it seems that a lipophilic moiety may be necessary to anchor the inositol head group for interaction with and activation of the channel, but any negatively charged groups mimicking the phosphate groups that can bind to the channel, such as CP1, will be able to activate the channel without the participation of an acyl chain. Alternatively, since externally applied CP1 could penetrate the membrane to interact with the site in the cytosolic domain of the channel . We found that CP1 application to ventricular myocytes increased IKs currents and reduced action potential duration  in which each compound has a formal charge of 2 (~104 compounds), targeting the PIP2 site 72 using the program MODELLER73. For ligand-based screening, by using a 3D molecular similarity calculation program ShaEP41, we ranked the compounds in the subset of ACD by their similarity scores with respect to the head group of PIP2. The best consensus compound, CP1, ranked in the top 2% for both structure-based screening (#197) and ligand-based screening (#34), was selected for experimental studies.A hybrid in silico screening strategy, combining both structure-based and ligand-based methods, was used in this study. For structure-based screening, by using an in-house molecular docking software MDockXenopus KCNQ1 channel (fKCNQ1) was solved using cryo-EM and published (PDB entry: 5vms)22. We then built a second homology model of hKCNQ1 based on the structure of fKCNQ1 59. Our modeled hKCNQ1 structure based on fKCNQ1 (PDB code: 5vms) was very close to this cryo-EM hKCNQ1 structure, with the backbone root-mean-square deviation (RMSD) of 1.5 Å. Hence, the use of the newly released cryo-EM human KCNQ1 structure for modeling does not change our docking results.In the interim of the study, the structure of the V4.2 (Q63881), NaV1.5 (Q14524), NaV β1 (Q07699), CaV1.2 (Q13936), CaV β1a (Q02641), CaVα2/δ1 (P54289), and HCN4 (Q9Y3Q4) were subcloned into oocyte expression vectors, respectively. Site-directed mutations of the KCNQ1 channel were all produced by overlap extension polymerase chain reaction (PCR) with high-fidelity Pfu polymerase . The presence of the desired mutation was verified by sequencing the PCR-amplified regions. All the primer sequences used in this study are available in Supplementary Table Complementary DNA (cDNA) encoding human KCNQ1 (UniProtKB/Swiss‐Prot under accession no. P51787), KCNQ2 (O43526), KCNQ3 (O43525), KCNE1 (P15382), Kir1.1 (P48048), hERG (Q12809), KXenopus laevis to obtain oocytes was approved by the Washington University Animal Studies Committee (protocol #20190030). Oocytes were manually dispersed and digested with collagenase  to remove the follicle cell layer. Stage V or VI oocytes were selected and injected with cRNAs (9.2 ng) of channel or mutations. Especially, to characterize IKs, a mix of cRNAs at a molar ratio 4:1 (KCNQ1:KCNE1) was injected. For KCNQ2/KCNQ3, the molar ratio was 1:1 (KCNQ2:KCNQ3). For sodium channels, a 3:1 molar ratio (β1:NaV1.5) and for calcium channels a weight ratio 1:1:1 (β1:α2/δ1:CaV1.2) was injected, respectively. Injected oocytes were incubated in ND96 solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.6, supplemented with 2.5 mM Na pyruvate and 1% penicillin–streptomycin, at 18 °C for 3–7 days before recording.The use of 75. Guinea pigs, weighing 300–500 g, were sacrificed by peritoneal injection of sodium pentobarbitone . The isolated cells were stored in KB solution containing (in mM) 83 KCl, 30 K2HPO4, 5 MgSO4, 5 Na pyruvate, 5 β-OH-butyric acid, 20 creatine, 20 taurine, 10 glucose, and 0.5 EGTA, pH 7.2.The use of guinea pigs was approved by the Stony Brook University Institutional Animal Care and Use Committee. Single ventricular myocytes were acutely enzymatically isolated from guinea pig heart as previously describedAll electrophysiological recordings on oocytes and isolated ventricular cells were performed at room temperature (20–23 °C).+ bath solution containing (in mM) 96 KCl, 4 NaCl, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.3–7.4. The HCN4 current was recorded in ND66 solution containing (in mM) 66 NaCl, 32 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.6. All the other potassium channels were recorded in ND96 solution. The currents of CaV1.2 channel were recorded in 40 mM Ba2+ bath solution containing (in mM) 40 Ba(OH)2, 50 TEA-OH, 2 KOH, and 5 HEPES, adjusted to pH 7.4 with methanesulfonic acid. CP1 (DISODIUM 5-BROMO-2- THIEN-2-YL]-1H-INDOL-3-YL SULFATE, Sigma-Aldrich, MO) and Chromanol 293B  were dissolved in bath solutions. All the chemicals were purchased from Sigma-Aldrich. To measure the I–V relations of the Kir1.1 channel, the test voltage was set from −100 to +60 mV for 1 s with 20-mV increments. The test pulses were stepped from a holding potential of 0 mV and then stepped back to 0 mV. The HCN4 currents were recorded by a test pulse between −30 and −140 mV in 10-mV step from a holding potential of −30 mV. For all the other potassium channels and CaV1.2 channel, the holding potential was −80 or −100 mV, and the test pulse was applied with 10-mV increment, which was followed with a repolarization pulse at −40 mV before returning to the holding potential. The durations of test and the repolarization pulse vary for different channels to allow the activation and deactivation of the channel to reach their steady states.The whole-oocyte ionic currents were recorded with a GeneClamp 500B amplifier  or iTEV90 amplifier  and then sampled at 1 kHz and low-pass-filtered at 2 kHz. The Patchmaster software  was used to acquire data. The Kir1.1 current was recorded with a high KV1.5 currents were recorded with a cut-open amplifier  coupled with an A/D converter . The data were acquired using Clampex software . The internal solution was composed of (mM) 105 NMG‐Mes, 10 Na‐Mes, 20 HEPES, and 2 EGTA, pH 7.4. The external solution was composed of (mM) 25 NMG‐Mes, 90 Na‐Mes, 20 HEPES, and 2 Ca‐Mes2, pH 7.4. The ionic currents were recorded using the standard I–V protocol. From a holding potential of −120 mV, cells were stepped to a 100-ms prepulse of −120 mV and then stepped to test potentials ranging from −120 to 60 mV with 20-mV increment, preceded by a 100-ms postpulse of −120 mV.Na+ solution . Then the cells were washed with ND96 solution and kept on ice until recording. A CA-1B amplifier was used to record whole-oocyte currents in ND96 solution. Fluorescent signals were recorded simultaneously using a Pin20A photodiode , filtered using a FITC filter cube , and then amplified using a patch-clamp amplifier .Oocytes were labeled on ice for 45 min with 10 μM Alexa 488 C5 maleimide  in high KXenopus oocytes were recorded from inside-out patches formed with borosilicate pipettes of 0.5–1.0-MΩ resistance. The data were acquired using an Axopatch 200-B patch-clamp amplifier (Axon Instruments) and Pulse acquisition software (HEKA). The pipette solution contained (in mM) 140 KMeSO3, 20 HEPES, 2 KCl, and 2 MgCl2, pH 7.2. The internal solution contained (in mM) 140 KMeSO3, 20 HEPES, 2 KCl, and 5 ethylene glycol tetraacetic acid (EGTA), pH 7.2.Macroscopic currents expressed in IKs current. The patch pipette solution contained (in mM) 140 KMeSO3, 20 HEPES, 2 KCl, 5 EGTA, and 5 MgATP, pH 7.4. The external Tyrode contains (in mM) 137.7 NaCl, 2.3 NaOH, 8 KCl, 1 MaCl2, 5 HEPES, 1 CdCl2, and 10 Glucose, pH 7.4. The cells were held at −40 mV and stepped to −20 mV, then to +60 mV with an increment of 10 mV, to measure the membrane current in the absence and the presence of chromanol 293B (10 μM). The chromanol 293B sensitive current was defined as the IKs current normalized to its cell capacitance. These experimental protocols and conditions eliminate most of the other membrane currents, such as IP, INa, ICa, and INa/Ca, except IK, so that we could obtain better experiment resolution.An Axopatch 1D amplifier  and the whole-cell patch-clamp technique were used to measure the 2, 1.8 CaCl2, 10 HEPES, and 10 Glucose, pH 7.4. The pipette solution is composed of (in mM) 115 K-aspartic acid, 35 KOH, 3 MgCl2, 10 HEPES, 11 EGTA, 5 Glucose, and 3 MgATP, pH 7.4.Action potentials were recorded with whole-cell patch-clamp recording techniques. Current-clamp configuration was used. Freshly isolated guinea pig cardiac myocytes were placed at 1 Hz with a stimulus of 180-pA amplitude and 10-mS duration. The APD was determined at 90% repolarization. Several minutes were allowed for the APD to reach steady state before data were collected. Typical Tyrode mimic AP Solution for action potential recording was used. The bath solution is composed of (in mM) 140 NaCl, 3 KCl, 1 MgCl27 proposed a five-state Markov model to conceptually recapitulate the gating process of KCNQ1 channels that involve two steps of VSD activation and two open states. We used this model to illustrate, in principle, that the CP1 effects on KCNQ1 channel gating can be produced by enhancing the VSD-pore coupling , β1 = c1 × exp(−v/n), αi = ai × exp(v/b), and βi = ci × exp(−v/d) (i = 2 and 3) are voltage-dependent transitions for the VSD activation, and k1–4 are rates (constant) for closed–open transitions, which describe the VSD-pore coupling. The values of the parameters in the simulations , and k1 = 2 (for +CP1), k2 = 853.08, k3 = 1 (for control), k3 = 4 (for +CP1), and k4 = 103.82. c3 was set to 0.090 ms−1 to balance the model. The output states for VSD movements are the combination of IC, IO, AC, and AO, and the output states for currents are the combination of IO and AO. Note that this is a simplified model without considering that one KCNQ1 channel is formed by four subunits. This model and the experimental data are not sufficient to describe the detailed mechanism of voltage-dependent gating or CP1 modulation of KCNQ1 channels.Our previous studiesG = G0 + 1/(1 + exp(V − V1/2)/S), where G0 is the minimum conductance, V1/2 is the half-maximal voltage of activation, and S is the slope factor. Dose–response curves in Fig. E = Emax/(1 − EC50/C)P, where EC50 is the drug concentration producing the half-maximum response and P is the Hill coefficient. In Fig. 50 values were obtained by fitting the data to IKs = IMin + (IMax − IMin) ([CP1]/([CP1] + EC50)) and V1/2 = V1/2 min + (V1/2Max − V1/2 min) ([CP1]/([CP1] + EC50)). The deactivation time course was fitted with single-exponential function. All data are expressed as mean ± SEM (n ≥ 3 or otherwise indicated). Electrophysiology experiments were performed on at least two separate batches of oocytes or myocytes to confirm reproducibility. The significance was estimated either using unpaired two-tailed Student t tests , Clampfit , and SigmaPlot . Normalized tail currents were plotted versus prepulse voltage and fitted with the Boltzmann function sts Fig.  or one-wsts Fig. . StatistFurther information on research design is available in the Supplementary InformationSupplementary Data 1Description of Additional Supplementary FilesPeer Review FileReporting Summary


We systematically analyzed the composition and morphology of FexCo1−xSe2/g-C3N4 and attributed the remarkable electrochemical performance of the catalyst to its unique structure. Fe0.2Co0.8Se2/g-C3N4 showed a superior HER activity, with quite low overpotential value (83 mV at −20 mA cm−2 in 0.5 M H2SO4) and a current density of −3.24, −7.84, −14.80, −30.12 mA cm−2 at 0 V (vs RHE) in Dulbecco’s Phosphate-Buffered Saline (DPBS), artificial sea water (ASW), 0.5 M H2SO4 and 1 M KOH, respectively. To the best of our knowledge, these are the highest reported current densities at this low potential value, showing intrinsic catalytic activity of the synthesized material. Also, the catalyst was found to deliver a high and stable current density of −1000 mA cm−2 at an overpotential of just 317 mV. Moreover, the synthesized catalyst delivered a constant current density of −30 mA cm−2 for 24 h without any noticeable change in potential at −0.2 V. These attributes confer our synthesized catalyst to be used for renewable fuel production and applications.To investigate cost affordable and robust HER and OER catalysts with significant low overpotentials, we have successfully embedded FeCoSe The fuel cell technology is considered as an efficient, long-lasting and environmental friendly source of energy. Among various existing forms of fuel cells, the H2/O2 fuel cells are deemed as zero carbon discharging technology with only water as the combustion product3. To ensure higher cell efficiency, the supply of pure fuel is essential and water electrolysis is one of the most convenient ways of producing H2. In fact, water splitting has garnered enough spotlight because of the highly pure and plentiful supply of fuel produced during this process. Likewise, this strategy does not demand elevated temperatures and pressure reactors. The overall water electrolysis can be explained by the following two half-cell reactions: hydrogen evolution reaction (HER) at the cathode5 and oxygen evolution reaction (OER) at the anode7, investigated in acidic and alkaline conditions, respectively8.Significant increase in the fossil fuel consumption and associated environmental threats of greenhouse gases have urged the scientific community to explore a sustainable and everlasting source of energy14 and Iridium or Ruthenium for OER18 being expensive and scarce, must be substituted by earth abundant and cost effective non-noble metals19 or metal free catalysts such as carbon based electrocatalysts. This work is an effort to substitute the expensive noble metals with only a small amount of earth abundant and cost effective non-noble metal catalysts to achieve better HER activity than the commercial catalysts reported so far. Among the metal free substrates, Carbon Nitride (C3N4) is the best choice as it offers π-conjugated graphitic planner layers with good conductivity and large surface area21. The C3N4 is reported as one of the primitive synthetic polymeric materials that offers different ways to modify its reactivity without any change in composition28. The g-C3N4 is believed to possess N-bridged poly(tri-s-triazine) planar structure with high degree of condensation. The s-triazine framework makes it thermally (up to 600 °C) and chemically stable. Moreover, it is a semiconducting material of medium band gap value (2.7 eV) that finds extensive use as a photo(electro)catalyst33.The noble metals i.e., Platinum for HER35. Likewise, various electrocatalysts including metal alloys37, metal nitrides39, metal phosphides40, metal carbides41, metal dichalcogenides42 and carbon-based catalysts have also attracted attention of researchers in the last few years44. The transition metal dichalcogenides (TMDs) have been found to be promising water splitting electrocatalysts and have thus gained significant interest in technological applications due to their unique electronic structure, different chemical compositions and diverse crystal symmetry. Several routes i.e. homogeneously dispersed electrocatalyst on conductive supports, chemical vapor and electron beam deposition have been developed to exploit the electrocatalytic properties of these materials47. However, the above-mentioned synthetic approaches are unfeasible for large scale production as control over the layers, chirality and influence of different substrates on its fabrication is still an area that is not well explored. The chemical doping is considered as an important structure-engineering technique to increase active sites and conductivity of these electrocatalysts48 which will be explored in this work.Recently, transition-metals sulfides, phosphides and selenides have been found to be efficient HER catalysts2, FeS2, and CoS2 displayed efficient overall water splitting performance51. Similarly, Iron (Fe) found in its natural form such as Nitrogenases and Hydrogenase  is the most promising material for HER. Hence, Iron doping is expected to increase the electrocatalytic performance of HER catalysts. Similarly, sulfur component of above-mentioned catalysts can be replaced with selenium. Sulfur and selenium are present in the same group and both exhibit similar properties, but selenides are expected to enhance HER performance of electrocatalysts due to low bond strength of Se–H (276 kJ/mol) than P–H (322 kJ/mol) and S–H (363 kJ/mol). Obviously, the bond strength has a great influence on the extent of adsorption/desorption of the intermediate states during catalysis. The weaker Se–H bond as compared to P–H and S–H is expected to facilitate the product desorption from catalytic sites which circumvents catalyst poisoning and thus stable and efficient performance is projected. Moreover, the Se sites can help to promote the delivery of oxygen molecules because of the localized negative charge of Se sites for OER. Likewise, there is a 3d–2p electronic repulsion amid metal and Se d-band centers32.Previously, Transition metal dichalcogenides (TMDs) such as NiS2 on graphitic carbon nitride substrate (FexCo1−xSe2/g-C3N4) as novel catalyst for overall water splitting. The catalytic activity of FexCo1−xSe2/g-C3N4 significantly improved with increased Fe percentages in the composite material. The Fe0.2Co0.8Se2/g-C3N4, has proven itself to be a promising electrocatalyst for HER and OER due to its high conductivity, unique structure and high surface area.Herein, we have reported a facile method for synthesizing highly active and stable FeCoSe3)3·9 H2O), Cobalt (II) Chloride hexahydrate (CoCl2.6H2O), Urea (CO(NH2)2) and Selenium Powder (Se) were purchased from Sigma-Aldrich. Potassium hydroxide, Sulfuric acid and Iso-propanol were of analytical grade and were used as received without any further purification. Preparation of Indian standard artificial sea water (ASW) and Dulbecco’s Phosphate-Buffered Saline (DPBS) is detailed in Supplementary note Iron (III) Nitrate  was kept very low and constant (0.8 mmol). For Fe0.2Co0.8Se2/g-C3N4, the molar ratio of Fe, Co, and Se was maintained at 1: 4: 10. Finally, feeding ratios of metals were further confirmed by average results of EDX (Energy Dispersive X-ray Spectroscopy).To synthesize CoSe2 and dried in air. These fabricated electrodes (with mass loading of 2.12 mg cm−2) were used as cathodes and anodes for respective electrochemical measurements of HER and OER respectively.3 mg of each sample and 20 μL of 5 wt.% Nafion were dispersed in 80 μL of iso-propanol to make a slurry. Then 5 μL of slurry was drop casted on glassy carbon electrode (GCE) of geometric surface area of 0.0707 cmTo identify the crystalline structure of products, X-ray diffraction (XRD) was performed on Rigaku D/max 2500 with a Cu Kα1 radiation source (λ = 1.54056 Å). Morphologies of prepared samples were identified at Hitachi S-4800 Scanning Electron Microscope (SEM) at an accelerating voltage of 30 KV. Energy dispersive X-ray spectra (EDX) were observed on an Oxford Material Analysis equipped on SEM and Transmission electron microscopy (TEM). While, Fourier Transform Infra-Red (FTIR) spectroscopic measurements were carried on TENSOR-27 (Bruker) and differential scanning calorimetry/thermal gravimetric analysis (DSC/TGA) on TA SDT Q600.2SO4, DPBS, ASW, and in 1 M KOH for HER using a three-electrode configuration at a scan rate of 20 mV/s. The FexCo1−xSe2/g-C3N4 powder on GCE was used as working, Platinum (Pt) wire as counter and Ag/AgCl (sat. KCl) as reference electrode. All potentials in this work are reported versus Reversible Hydrogen electrode (RHE) according to the Eq. Electrochemical studies were carried out on Autolab PGSTAT302N Metrohm, (running with NOVA 1.11 software) electrochemical workstation at room temperature. While, EIS measurement was made with a Gamry EIS 300 electrochemical impedance analyzer with frequencies ranging from 100 kHz to 0.1 Hz at a voltage amplitude of 10 mV. The electrocatalytic ability of all materials was studied in 0.5 M H2 (JCPDS # 53- 0449) and FeSe2 (JCPDS # 21- 0432), respectively. It is noteworthy that the diffraction peaks of g-C3N4 are not indexable in the Fe0.2Co0.8Se2/g-C3N4 partly due to their comparatively low diffraction intensity, as carbon nitride synthesized from urea shows low diffraction intensity, and partly to relatively small amount of carbon nitride present in the sample. In addition, with increase in Fe amount, the intensity of XRD signals decreased which implied low crystallinity of the Fe0.2Co0.8Se2/g-C3N4 material in comparison to the pristine ones. Furthermore, the final product (Fe0.2Co0.8Se2/g-C3N4) showed diffraction peaks corresponding to both CoSe2, and FeSe2, which indicates the coexistence of the two orthorhombic phases52. It is pertinent to mention here that owing to the small differences in ionic radii of iron and cobalt, the strongest diffraction peaks of FeCoSe2, FeSe2 and CoSe2 displayed very little interlayer spacing difference (±0.01 Å), which provided the basis for facile doping of Fe into the CoSe2 structures. Additionally, Fourier transform infrared spectroscopy (FTIR) was conducted to reveal the bonding states of FexCo1−xSe2/g-C3N4 samples  technique and is shown in Fig. les Fig. .Figure 12/g-C3N4) exhibited a not so well-defined nodular morphology  to study the effect of temperature, phase transition phenomenon and the associated endothermic or exothermic effects Figs.  and S5. xCo1−xSe2/g-C3N4  were investigated in 0.5 M H2SO4, DPBS, ASW and 1 M KOH. The best electrocatalytic performance was observed for Fe0.2Co0.8Se2/g-C3N4. Fig. 2SO4. The Fe0.2Co0.8Se2/g-C3N4 showed best electrocatalytic activity with highest current density values of −14.8, −20, −100 mA cm−2 at potential values of 0, 83, and 209 m V respectively in 0.5 M H2SO4 solution. As HER is facilitated in acidic medium, the current density of −14.8 mA cm−2 is likely due to the formation of HSeO3− in strong acidic conditions and its subsequent reduction to H2Se and or HSe− 53. To further explore the role of acid, HER was also carried out in DPBS solution where FeCoSe2 exhibited a low current density value of −3.24 mA cm−2, confirming the role of acid in the formation and reduction of HSeO3− . However, with further increment of the Fe content after optimal ratio (20 wt%), the HER activity sharply decreased due to its probable changes in electronic configurations. In the same way, the electrocatalytic ability of the Fe0.2Co0.8Se2/g-C3N4 was also assessed in DPBS, ASW, 0.1 M H2SO4 and 1 M KOH aqueous solutions as shown in Fig. −2 in 1 M KOH is probably due to a reduction contribution of SeO32− species that are generated at the catalyst-electrolyte interface during testing at high pH conditions. The overall performance of Fe0.2Co0.8Se2/g-C3N4 is comparable to the benchmark HER electrocatalysts  in comparison with CoSe2/g-C3N4, as the arc diameter of FeCoSe2 is smaller than CoSe2 dispersed on substrate. Thus, the EIS of Fe0.2Co0.8Se2/g-C3N4 was further performed at different applied potentials. It can be seen from Fig. CT values decrease with increase in applied potential value. Therefore, further increase in applied potential i.e. −0.295 V decreases the RCT and hence enhances the HER activity. Moreover, determining the electrocatalytic active surface area (ECSA) is an important parameter since it shows the intrinsic performance of electrocatalysts. ECSA was calculated by double-layer capacitance (Cdl) value  of CoSees Table .0.2Co0.8Se2/g-C3N4, chronopotentiometric response was recorded at constant current density of −30 mA cm−2 ,  was investigated in aqueous 1 M KOH solution. Feles Fig. . To acqules Fig.  and S11.2 hybrid uniformly supported on g-C3N4 that performed efficiently as a bifunctional electrocatalyst. Introduction of iron was found to be of vital importance as it led to increase in TOFmin and electrocatalytic ability of nanohybrid material. Due to optimized Fe incorporation, presence of Se atom and a good conducting substrate, the Fe0.2Co0.8Se2/g-C3N4 ensured high ECSAs and enhanced electrocatalytic performance in the context of low overpotential, small Tafel slope, high current density, high proton conductivity, and stability. In addition, the designed catalyst also performed good OER activity with moderate onset potential and high current density values. Due to facile synthesis technique and good electronic properties, g-C3N4 can offer a perfect platform for the conversion and modification of catalysts with varied structure and composition. In this work, g-C3N4 allowed the catalyst to work in wide pH ranges which is highly desirable for industrial applications. These peculiar qualities imply that Fe0.2Co0.8Se2/g-C3N4 hybrid electrocatalyst has the potential to replace benchmark electrocatalysts for clean fuel production in industrial applications.A low cost and a facile hydrothermal route was adopted for the preparation of FeCoSeSupplementary Information.


Agave spp. plants that are cooked and pressed. Agave must contains a high amount of fructose and phenolic compounds, and fermentation usually occurs under stressful (and uncontrolled) environmental conditions. Yeasts capable of growing under such conditions usually display advantageous biological and industrial traits for stress tolerance such as flocculation. In this study, seven Saccharomyces cerevisiae strains isolated from mezcal must were exposed to temperatures ranging between 10 and 40 °C, and to different sugar sources (fructose or glucose). Yeasts grown in fructose increased their stress tolerance, determined by colony count in a microdrop assay, under low temperature (10 °C) compared to the growth at 40 °C on solid cultures. The most stress-tolerant mezcal strain (Sc3Y8) and a commercial wine (Fermichamp) strain, used as control, were grown under fermentation conditions and exposed to long-term temperature stress to determine their performance and their potential for flocculation. Compared to glucose, fermentation on fructose increased the metabolite accumulation at the end of culture, particularly at 40 °C, with 2.3, 1.3 and 3.4 times more glycerol (8.6 g/L), ethanol (43.6 g/L) and acetic acid (7.3 g/L), respectively. Using confocal microscopy analysis, we detected morphological changes such as aggregation and wall recognition at the level of budding scars in yeast, particularly in the Sc3Y8 strain when it was exposed to 40 °C. The analysis confirmed that this mezcal strain was positive for flocculation in the presence of Ca2+ ions. Analysis of FLO1, FLO5 and FLO11 gene expression implicated in flocculation in both Saccharomyces strains showed a strong transcriptional induction, mainly of the FLO5 gene in the mezcal Sc3Y8 strain.Mezcal is a distillate produced by spontaneous fermentation of the must obtained from stalks of  Before each assay, a loop of each strain was pre-cultured in 250-mL Erlenmeyer flask with 50 mL of YPD broth  for 18 h at 30 °C and 200 rpm in an incubator .Six yeast strains belonging to our laboratory culture collection , obtained from fermenting mezcal must, were cultivated in yeast extract peptone dextrose (YPD) agar  (Two culture media were used on yeast extract peptone (YP) plates  with 20 g/L of either glucose (YPD) or fructose , to evaluate the influence of the carbon source on the stress tolerance of these yeasts. Selection of thermotolerant yeasts was conducted using a microdrop assay  capped with cotton stoppers and maintained for 60 h without agitation at 30 or 40 °C. Cell population was determined by Neubauer chamber counting at different time intervals. Experiments were performed in triplicate.Stress-tolerant yeasts were cultured in YP broth  supplemented with 100 g/L of either glucose (YPD) or fructose . An inoculum concentration of 1.5‧102SO4 (5 mM) in Milli-Q water  for 30 min, and coupled to a refractive index (RI) detector. The samples were centrifuged at 14 400xg for 5 min at 10 °C in a Spectrafuge 24D , then diluted  in the mobile phase and filtered through nylon membranes . Calibration curves within ranges of 0-20 (ethanol), 0-10 (glycerol) and 0-10 g/L (acetic acid) were set up for commercial standards . Finally, experiments were performed in triplicate.Residual reducing sugars in YPD or YPF broth were detected colorimetrically by a modified dinitrosalicylic acid (DNS) method  and the number of cells of each strain present in a Neubauer chamber. Each experiment was performed in triplicate. Linear regressions and their fitting were: y=2‧10Flocculation was determined using the microflocculation method  and then 0.4% (m/V) Solophenyl Flavine 7GFE 500  was added. Incubation was conducted at room temperature in darkness. The samples were washed with citrate buffer. Images were visualized and acquired with a confocal microscopy TCS SP5 X  at magnifications 20× and 40× with and without 4.0 digital zoom  at 490 and 510-550 nm for excitation and emission, respectively. Finally, deionized water instead of the citrate buffer served as a negative flocculent control.Flocculation was analyzed by confocal microscopy. Briefly, selected flocculent and non-flocculent strains were grown at 30 and 40 °C as described above. ScFerm was selected as the non-flocculent yeast. Cells were resuspended in citrate buffer  cells grown in YPD broth using the DNAzol kit . DNA amount and quality were determined by using NANODROP 2000 equipment (Thermo Scientific). DNA samples were adjusted at final concentration of 100 ng/μL. For real-time polymerase chain reaction (PCR) amplification of FLO1 and FLO11 genes, the following primer sets were used: FLO1-F 5´-ATGCCTCATCGCTATATGTTTTTG-3´ and FLO1-R 5´-GCTCCTGAGGCCACACTAGTTAG-3, and FLO11-F 5´-CCTCCGAAGGAACTAGCTGTAATT-3´ and FLO11-R 5´- AGTCACATCCAAAGTATACTGCATGAT-3´, which produce fragments of 68 and 103 bp, respectively  from S. cerevisiae S288c (NM_001179342.1) sequence. Then, using this FLO5 sequence, a new primer set was designed (FLO5-F2 5’-GCATCAGGAAGTACGGAAGTCA-3’ and FLO5-R2 5’-TGCTGCATTCGAATATGTGGA-3’), which corresponds to a 110-bp fragment. MEGA v. 4.1 , 2 µL dNTP mix , 2.5 µL PCR buffer (Bioline), 1.5 µL each primer (50 ng), 0.2 U Taq polymerase (Bioline), and 3 µL DNA template (100 ng). Initial denaturalization time was 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 61 °C and 1 min at 72 °C, with a final extension cycle of 10 min at 72 °C. PCR products were verified and purified from an agarose gel (1%) using Wizard SV gel and PCR clean-up kit . These PCR products were then bound and cloned in a pGEM-T vector (Promega) and transformed using Escherichia coli DH10B competent cells . FLO1, FLO5 and FLO11 gene identity was confirmed by sequencing with an ABI 377 DNA sequencer equipment  with universal M13 primers and the BigDye terminator v. 3.1 cycle sequencing kit (Applied Biosystems).Genomic DNA isolation was conducted in FLO1, FLO5 and FLO11 genes was determined on selected (Sc3Y8 and ScFerm) strains grown on YPD broth at 30 and 40 °C. Cells were collected after 12, 24, 48, 60 and 72 h. RNA isolation was carried out using the TRI reagent kit (Molecular Research Center) according to manufacturer’s instructions. RNA (1 µg) was treated with 1 U RQ1 RNase-Free DNase I (Promega), adjusting the volume to 10 µL, and incubated for 30 min at 37 °C. Reaction was stopped by adding 1 µL RQ1 DNase Stop Solution (Promega) followed by an incubation for 10 min at 65 °C. Genomic DNA contamination was verified by using agarose gels at 1.3% and by PCR reaction as follows: RNA treated with DNase was used as negative amplification template for the UBC6 gene. This encodes a constitutive ligase EI protein involved in the ubiquitin-protein degradation , 2 µL dNTP mix , 2.5 µL PCR buffer (Bioline), 1.5 µL each primer (50 ng), 0.3 µL Taq polymerase (Bioline), and 3 µL genomic DNA template (100 ng) or RNA treated with DNase (50 ng). Initial denaturalization time was 5 min at 94 °C, followed by 35 cycles at 94 °C for 1 min, 1 min at 59 °C and 1 min at 72 °C, with a final extension cycle at 72 °C for 10 min. cDNA synthesis was carried out using 1 µg RNA and the GoScriptTM Reverse Transcription System (Promega). Briefly, a final reaction volume of 20 µL containing 1 μg of DNase I‐treated RNA was mixed with 0.5 μg Oligo(dT)15 and random primers. Then, the reaction was incubated at 70 °C for 5 min, chilled in ice, and an RT Master Mix containing GoScript™ 1× reaction buffer, 5 mM MgCl2, 0.5 mM each dNTP, 1 U Recombinant RNasin Ribonuclease Inhibitor and 15 U/μg GoScript™ Reverse Transcriptase was added. Reaction was incubated at 25 °C for 5 min, then at 37 °C for 1 h, and finally at 70 °C for 15 min. A kanamycin-positive control of RNA provided in the kit was used to estimate the yield of cDNA synthesis. cDNA amount was verified in a NANODROP equipment  and kept at -60 °C until its use.Expression of the FLO genes was determined by the ΔΔCq method (UBC6 gene as a reference (Expression of Gene relative expression (R) was calculated using the following equation:Ref and ETarget are the efficiencies of the reference and target gene, respectively, and CtRef and CtTarget are the threshold cycle (Ct) average values of the reference and target gene, respectively  showed that the carbon source affected the growth behaviour since all the eratures , but onlSaccharomyces strains had a lower growth than at 30 °C. Regarding sugar consumption, these strains show different profiles since only the ScFerm completely consumed both sugars at 30 °C (after 30 h), but left 50 and 23 g/L of glucose and fructose, respectively, at 40 °C. The Sc3Y8 strain had a high residual sugar concentration of 17 and 31 g/L of glucose and fructose, respectively, at 30 °C, and overall it was less efficient than the industrial strain at this temperature. At 40 °C the residual sugar concentration was similar to the one of ScFerm. Different fructose uptake at different temperatures has been previously reported by Dumont , which is known to ferment grape must sugars to dryness . InteresS. cerevisiae strains (S. cerevisiae strains (etc.) conditions, Saccharomyces strains tend to overproduce glycerol and acetic acid, and to produce less biomass, as corroborated with the colony count in i.e. the number of viable cells that grew in glucose or fructose at the two highest temperatures tested for all the strains. This general trend can be observed for both hexoses when comparing metabolite profiles , glycerol, ethanol and acetic acid concentrations increased in all selected strains when grown on fructose at 40 °C . Sc3Y8 sprofiles  at 30 anS. cerevisiae, as a mechanism to overcome stress conditions  . Confocal microscopy analysis of Sc3Y8 strain using the fluorophore Solophenyl Flavine 7GFE 500  allowed to assess some changes in the cell wall conformation at 40 °C, similarly to the observations made by Soares  . This ph budding . In cont budding . To conf2 showed cell aggregates only in the Sc3Y8 strain. These cell aggregates showed apparent different sizes depending on the temperature conditions (data not shown). Finally, at 40 °C, less lysed cells were observed in the Sc3Y8 than in ScFerm strain, supporting the previous viability assay. These results suggested that the flocculation phenotype in the Sc3Y8 strain may contribute to its thermotolerance due to the changes in the cell wall conformation and integrity, allowing a higher viability. Also, it has been reported that temperature affects the flocculation phenomenon at the level of cellular interactions, probably as a consequence of the flocculin denaturalization (S. cerevisiae (F6789A) strain after deletion of the FLO5 gene. These genes regulate stress tolerance and are related with cell wall reorganization and adhesion . FLO1, FLO5 and FLO11 transcript accumulation was analyzed in these Saccharomyces strains on YPD broth at 30 and 40 °C. Results showed that FLO1 gene transcripts were constant at 30 °C in both Saccharomyces strains. However, at 40 °C a slight but significant  induction (6%) was observed after 12 h, particularly in the Sc3Y8 strain. ScFerm also exhibited a reduced induction (3.8%) at 60 h of culture (FLO5 expression in Sc3Y8 strain was induced at 40 (up to 20-fold) than at 30 °C. This transcript induction was statistically different among strains after 12 h, and had a maximal value of 175% at 48 h in the Sc3Y8 strain, which coincides with the beginning of the stationary phase  conditions in different Saccharomyces wine strains (S. cerevisiae wine strain (F6789A-Δ flo5) causing an up-regulation of genes related to assembly, shape and cell adhesion, and genes related to the organization of the cell wall, among others . At 40 °C, there was a constantly low expression of FLO11 in both strains. This gene has been related to a wide phenotype variety, such as flocculation, the invasive growth and ﬂor formation  industrial strain. Flocculation seems to be associated with morphological cell wall changes that might contribute to its thermotolerant phenotype and to a higher viability, which in turn allows a better fermentation productivity at high temperature. Nonetheless, to corroborate this relation, the construction of FLO-specific mutants is required to address their exact role in the thermotolerance observed in the flocculating strains. Indeed, more effort must be made to clarify these technologically important physiological traits.


C) is a high-affinity receptor for Aβ oligomers and mediates some of their toxic effects. The N-terminal region of PrPC can interact with Aβ, particularly the region encompassing residues 95–110. In this study, we identified a soluble and unstructured prion-derived peptide (PrP107–120) that is external to this region of the sequence and was found to successfully reduce the mitochondrial impairment, intracellular ROS generation and cytosolic Ca2+ uptake induced by oligomeric Aβ42 ADDLs in neuroblastoma SH-SY5Y cells. PrP107–120 was also found to rescue SH-SY5Y cells from Aβ42 ADDL internalization. The peptide did not change the structure and aggregation pathway of Aβ42 ADDLs, did not show co-localization with Aβ42 ADDLs in the cells and showed a partial colocalization with the endogenous cellular PrPC. As a sequence region that is not involved in Aβ binding but in PrP self-recognition, the peptide was suggested to protect against the toxicity of Aβ42 oligomers by interfering with cellular PrPC and/or activating a signaling that protected the cells. These results strongly suggest that PrP107–120 has therapeutic potential for AD.Alzheimer’s disease (AD) is the most prevalent form of dementia and soluble amyloid β (Aβ) oligomers are thought to play a critical role in AD pathogenesis. Cellular prion protein (PrP Alzheimer’s Disease International (ADI) and the American Alzheimer’s Association (AA) have estimated that over than 50 million people are living with dementia, and that Alzheimer’s disease (AD) is the most common cause and may account for 60–70% of dementia cases . AccordiEvidence indicates that AD is an aging-related disease, and frequency in individuals aged 85 or older is higher (one in three) compared to age 65 (one in nine) ,2. AD inAlthough accumulation of senile plaques formed by Aβ is a major histopathological trait of AD, it is widely accepted that soluble Aβ oligomers, forming as intermediate species in the process of neuritic plaque formation or released from the plaques, can effectively play a key role in neuronal dysfunction and impair synaptic structure and function ,7. It isC mediates the abnormal effects of Aβ oligomers, particularly the oligomer-induced inhibition of LTP [C [C [C and Aβ reduces longevity in Drosophila melanogaster, and that expression of Aβ individually cannot create pathogenic phenotypes [C mediates the aberrant effects of Aβ oligomers, other studies have indicated that it is not a necessary component for the toxicity cascade induced by Aβ oligomers, as Aβ-induced LTP inhibition or memory impairment has also been reported to occur independently of the overexpression or ablation of PrPC in the transgenic mice [Several studies have reported that the cell membrane protein PrPn of LTP ,14,15. If LTP [C , and thaTP [C [C . Other renotypes . In spitnic mice ,20,21. Tnic mice .C is a membrane protein that is found in both neurons and glial cells [C consists of a single polypeptide chain of 208 amino acid residues (residues 23–230). The N-terminal region of the protein (residue 23–120) is unstructured, while the C-terminal region (residues 121–230) is largely structured, consisting of three α-helices  and two short anti-parallel β-strands  [C function is poorly understood, although roles have been suggested in synaptic transmission, long-term memory, circadian rhythms, T cell function, hematopoietic stem cell renewal, copper-binding, apoptosis and oxidative stress homeostasis, among others [Sc) is derived from PrPC [Sc) can cause a group of neurodegenerative diseases affecting both human and animals [PrPal cells  and has al cells ,25. The 161–164) . The pro161–164) ,27,28. Pg others . The scrrom PrPC . The cen animals .C has a high binding affinity for Aβ42 oligomers [C-expressing and PrPC knock-out mice were equally susceptible to Aβ42 oligomer-induced cognitive impairment recognized that the oligomers interacted with PrPC with high affinity [42 oligomers and PrPC dates back to 2009, when it was also found that the N-terminal region of PrPC, particularly the 95–110 residues, was involved in the binding [N-terminal residues 23–27 and 92–110 were critically important for binding [A large number of data have shown that PrPligomers ,34. Evenaffinity . The fir binding . One yea binding . The imp binding ,40,41,42N-terminal region of PrPC in the binding of Aβ oligomers, none of the studies reported so far have highlighted a role of the region encompassing residues 106–126 in Aβ42 oligomer binding [C-PrPSc interaction [Unlike the many reports showing a role of the  binding . By cont binding ,47,48,49eraction ,45, and eraction .C complex formation, we reasoned that a short peptide encompassing this region of the sequence might inhibit Aβ42 oligomer toxicity. The hypothesis for this idea is that such a peptide might leave the Aβ oligomers unbound and unaltered, while engaging in interactions with PrPC. Our designed peptide encompassing residues 107–120 (PrP107–120) was found to be very soluble in physiological conditions, most probably because it lacks the highly hydrophobic region VVGGLG (residues 121–126) of the PrPC fragment 106–126 responsible for prion aggregation [42 oligomers on neuroblastoma SH-SY5Y cells in absence of Aβ42 oligomer binding and structural reorganization, but in presence of partial binding between PrP107–120 peptide and endogenous cellular PrPC. Since this peptide is not aggregation-prone and has beneficial effects against Aβ-induced toxicity, it is of potentially great interest for rationalizing the pathogenesis of AD and routes to its prevention, as well as in setting up therapeutic strategies for the treatment of AD.As a segment involved in PrP self-recognition, but not in Aβ-PrPregation ,45,49. Ih) for a peptide with the size of PrP107–120 (14 amino acid residues) in an unfolded state, using the equation described in h value was found to be 0.99 ± 0.56 nm. Dynamic light scattering (DLS) shows that PrP107–120 dissolved in water has a hydrodynamic diameter (Dh) of 2.11 ± 0.68 nm, corresponding to a Rh of 1.05 ± 0.34 nm  under these conditions, which appears dominant in the population if we take into account that the intensity of scattered light scales with the sixth power of the diameter  formed from Aβ42 peptide according to a well-established protocol [42 oligomers because they are widely used [2+ and reactive oxygen species (ROS) levels in cultured cells [42 ADDLs with a final concentration of 3-µM monomer equivalents (m.e.) on the metabolic activities of human SH-SY5Y cells. This immortalized neuronal cell model is mostly used for AD research, as human cholinergic neurons are difficult to obtain and maintain and unsuitable for routine experiments.In order to analyze whether PrPprotocol . ADDLs wely used ,53,54,55ely used ,57. Thesed cells , and haved cells . To this42 ADDLs at 3 µM m.e.  to Aβ42 ADDLs (3 µM) significantly increased cell viability to 79.7 ± 4.3% compared with the cells treated with Aβ42 ADDLs (3 µM) in the absence of the peptide . In this case, we found a cell viability of 86.4 ± 2.5%, again indicating protection by the PrP107–120 peptide. These results suggest that PrP107–120 can act as an inhibitor of Aβ42 ADDL oligomer toxicity.The metabolic activity of the cells was analyzed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay, which is a widely used indicator of mitochondrial reduction capacity. The ability of SH-SY5Y cells to reduce MTT significantly decreased to 66.6 ± 4.5% following treatment for 24 h with Aβ µM m.e. . By contectively . The add peptide . In anot42 ADDLs, and Aβ42 oligomers more generally, when added to the cellular medium, is the permeabilization of the plasma membrane with a rapid influx of calcium ions (Ca2+) from the extracellular space to the cytosol [107–120 can prevent the increase of Ca2+ levels mediated by the Aβ42 ADDL oligomers, we monitored the influx of Ca2+ in SH-SY5Y cells treated with Aβ42 ADDLs in presence and absence of PrP107–120, at the same concentrations used for the MTT assay. The quantification of the intracellular Ca2+-derived fluorescence in confocal microscopy images shows that the treatment of the cells with Aβ42 ADDLs caused a significant increase in intracellular Ca2+ up to 246 ± 21% compared with untreated cells, taken as 100%  that was significantly lower than that observed in cells treated with Aβ42 ADDLs .In order to assess whether the protective role of PrP of Aβ42 ,63,64,65107-120 on the cellular internalization of Aβ42 ADDLs in SH-SY5Y cells, we performed immunostaining experiments using the 6E10 specific antibody against Aβ42 and wheat germ agglutinin to stain Aβ42 and the cell membrane, respectively. Cells were exposed for 1 h to the various protein samples described above and added to the cell medium. Thus, for the intracellular Aβ42-derived fluorescence (green) in the SH-SY5Y cells, we observed more than 78% reduction in cells treated with Aβ42 ADDLs + PrP107–120, and more than 74% reduction in cells treated with Aβ42 + PrP107–120 in terms of intracellular ADDLs levels with respect to cells treated with Aβ42 ADDLs taken as 100% , we carried out a number of tests using dot-blot, ThT fluorescence, far-UV CD and ANS fluorescence on ADDLs in the presence and absence of PrP107–120, using the same samples used for cell toxicity.To determine whether PrP42 ADDLs was monitored by a dot-blot immunoassay using the conformation-sensitive antibody 19.3 specific for Aβ42 ADDLs [42 species. For Aβ42 ADDLs, Aβ42 + PrP107–120 and Aβ42 ADDLs + PrP107–120 we observed a recognition for both antibodies, whereas the PrP107–120 spot did not show any cross-reaction  or pre-incubated in the presence of the peptide after their formation (Aβ42 ADDLs + PrP107–120), suggesting again that the PrP107–120 peptide was not able to change the Aβ42 ADDL structure  PrP107-120 binds to ADDLs and shields their hydrophobic patches that are responsible for toxicity, or (ii) it interacts with the cells and renders them less vulnerable to ADDL toxicity. To verify which hypothesis was correct, the interaction of PrP107–120 and Aβ42 was studied in cell cultures. Confocal microscopy images showed an absence of co-localization between Aβ42 ADDLs (3 µM m.e.) and PrP107-120, both with ratios of 4:1 (0.75 µM PrP107–120) and 1:1 (3 µM PrP107–120), suggesting an absence of interaction between the two peptides (107–120 (0.75 µM) was found to partially colocalize with endogenous cellular PrPC  and a synthetic peptide corresponding to residues 98–107 (PrP98–107) were found to inhibit the toxicity of the Aβ42 ADDLs to organotypic hippocampal slices, whereas a control PrP213–230 peptide did not have any such effects [98–107 peptide exert their effect by binding to PrPC and the diffusible Aβ42 oligomers, respectively, thus preventing their interaction in both cases [Previous studies have suggested that the cell surface protein PrPunctions ,16,17,18unctions ,20,21. Os 95–110 ,40,41,42 effects . It was th cases .C peptide binding to Aβ42, we have used a synthetic PrPC peptide involved in PrP self-recognition (PrP107–120), with the goal of achieving a similar biological result with a different mechanism, namely, binding to membrane-anchored PrPC and impeding the binding of the latter to Aβ42 oligomers. We found that PrP107–120 significantly reduced the toxicity of Aβ42 oligomers with or without pre-incubation of the peptide with Aβ42 during the process of ADDL oligomer formation. Biophysical analyses and a dot-blot immunoassay excluded an alteration of the Aβ42 ADDL structure or aggregation state in the presence of the peptide, ruling out the hypothesis that the reduction of Aβ42 oligomer toxicity and internalization mediated by PrP107-120 is due to this reason. Moreover, no PrP107–120 colocalization with Aβ42 was found in the cell cultures using confocal microscopy even at high concentrations of PrP107–120, indicating a lack of any significant interaction between the two peptide species. In contrast, a partial colocalization between PrP107-120 and endogenous PrPC was found, indicating that the peptide can bind to the cellular prion protein. Furthermore, akin to previous observations, the role of PrPC in mediating oligomer toxicity is not restricted to Aβ42 oligomers, but also to other β-sheet rich proteins [107–120 on Aβ42 oligomers and type-A HypF-N oligomers, used here as a positive control of toxic oligomeric species. However, we cannot completely exclude that PrP107–120 stimulates a well-defined intracellular signaling [42 oligomer toxicity independently of its interaction with membrane-anchored PrPC.In this study we have used a different perspective: Rather than using a synthetic PrPproteins , and ourignaling  and causC involved in self-recognition, or other molecules potentially mimicking the same sequence trait, for the treatment of AD. This could open new avenues to the identification of the mechanism of interaction of soluble prion-derived peptides and Aβ42 oligomers, as well as to the mechanism through which PrPC mediates the toxic effects of Aβ42 oligomers.Overall, although the results obtained here in vitro on a cell culture line need to be validated in vivo on animal models, the present study shows the potential therapeutic value of peptides corresponding to the region of the sequence of PrP107–120) with the sequence Ac-TNMKHMAGAAAAGA (purity by HPLC > 95%) was purchased from Biomatik . The <5% impurities are mainly peptides with similar sequences, as are often found in solid-state peptide synthesis [The synthetic human prion protein fragment spanning residues 107–120  was dissolved in pure hexafluoro-2-isopropanol (HFIP) to 1 mM. For each experiment, the solvent was evaporated using gentle nitrogen flow and the peptide was reconstituted in 2% (v/v) dimethyl sulfoxide (DMSO) and F12 Ham medium to a final concentration of 100 µM. Aβ-derived diffusible ligands (ADDLs) were prepared after 24 h incubation at 4 °C, as previously reported [42 ADDLs; (B) 25 µM PrP107–120  added to preformed 100 µM m.e. of Aβ42 ADDLs at a 1:4 molar ratio, incubated at 4 °C for 2 h (Aβ42 ADDLs + PrP107–120); (C) 25 µM PrP107–120  added to 100 µM Aβ42 at a 1:4 molar ratio before Aβ42 aggregation, maintained at 4 °C for 24 h under the same conditions used to form Aβ42 ADDLs (Aβ42 + PrP107–120); and (D) 25 µM PrP107–120. All four samples were in 2% (v/v) DMSO and F-12 Ham medium and were diluted 33-fold before each experiment into cellular medium without DMSO to final concentrations of 3 and 0.75 µM (m.e.) for Aβ42 and PrP107–120, respectively.Lyophilized synthetic Aβreported , and chereported ,57. In tg) for 10 min, and the concentration was measured at 280 nm. The sample was then diluted to 48 µM in 12% (v/v) trifluoroethanol, 50 mM acetate buffer and 2 mM dithiothreitol, at pH 5.5, as this condition is known to promote aggregation of HypF-N and proteins of the same structural family [g) for 15 min and the pellet was dried with a gentle nitrogen flow and resuspended to 12 µM m.e. in cellular medium with or without PrP107–120, to a final concentration of 3 µM (4:1 molar ratio).HypF-N was purified as described previously  and storl family ,69. Aftew/v CuSO4·5H2O in water). The blank and protein samples were mixed with the resulting BCA working reagent at a 1:8 ratio; thus, 25 µL of each peptide or BSA or blank samples were mixed with 200 µL of BCA working reagent and were added to 96-microplate wells. The plate was incubated at 60 °C for 15 min and, after 5 min cooling to room temperature, the absorbance of all wells was measured at 562 nm using an ultrafast BioTek Synergy H1 plate reader . All absorbance values were blank subtracted. Peptide concentration was calculated by interpolation using a standard curve (absorbance versus protein concentration) obtained with the eight BSA samples.Stock bovine serum albumin (BSA) standard  was prepared at a 2-mg/mL final concentration in water. Eight BSA samples with concentrations ranging from 0 to 2000 µg/mL were prepared by dilution for the standard curve. The bicinchoninic acid (BCA) working reagent was prepared by mixing 10 mL of solution A  with 200 µL of solution B (4% 107–120 was investigated under different solution conditions at 37 °C: (A) 0.5 mg/mL peptide in 20 mM phosphate buffer, 200 mM NaCl, pH 7.0; (B) 0.5 mg/mL peptide in 20 mM HCO3–, 200 mM NaCl, pH 10.5; (C) 0.5 mg/mL peptide in 20 mM phosphate buffer, 200 mM NaCl, 10% (v/v) trifluoroethanol, pH 7.0; (D) 1.0 mg/mL peptide in 20 mM acetate buffer, 200 mM NaCl, pH 4.0; (E) 1.0 mg/mL peptide in 20 mM HCO3–, 200 mM NaCl, pH 10.5; (F) 1.0 mg/mL peptide in 20 mM phosphate buffer, 500 mM NaCl, pH 7.0; (G) 1.0 mg/mL peptide in 20 mM phosphate buffer, 15% (v/v) methanol, pH 7.0; (H) 1.0 mg/mL peptide in 20 mM phosphate buffer, 200 mM Na2SO4, pH 7.0. Fibril formation was monitored by the ThT fluorescence assay, DLS and CD spectroscopy for all conditions.The aggregation kinetics of PrP107–120, 1 mg of peptide was dissolved in 1 mL of water. The sample was filtered through a 0.22-µm filter and size distribution analysis was performed at 25 °C by a Zetasizer Nano S DLS device from Malvern Panalytical  thermostated with a Peltier system and checked for its reliability with polystyrene latex beads with known hydrodynamic diameter. A 10-mm plastic cell with a reduced volume was used. The refractive index and viscosity were 1.333 and 0.88 cP. The cell position and attenuator index were set automatically. The theoretical Rh for a peptide was calculated by the following equation:h was twice the Rh value. In a second experiment, to investigate the effect of different conditions on peptide aggregation, measurements were carried out using 1 mg of peptide dissolved in 1 mL of selected buffers and following the size distribution over time for several days at 37 °C, using the same technical apparatus and cell described above. The refractive index and viscosity were set according to the various conditions.In a first experiment, to determine the size of monomeric PrPresidues . The theThT (Sigma-Aldrich) was dissolved to 25 µM, in 25 mM phosphate buffer, with a pH of 6.0, then filtered by a 0.45-µm filter. For a given peptide sample, 60 µL aliquots were added to 440 µL of resulting solution. A 2 × 10 mm optical path-length cuvette was used, and fluorescence spectra were recorded at 37 °C using a PerkinElmer LS 55 fluorimeter  equipped with a thermostated cell holder attached to a Thermo Haake C25P water bath  with an excitation wavelength of 440 nm and an emission wavelength range of 460–600 nm.107-120 polymerization kinetics in the presence of different conditions, 0.2 mg/mL of PrP107-120 peptide was used. To investigate the effect of the PrP107–120 peptide on Aβ42 oligomeric ADDLs, three samples were prepared, each 300 µL, including two separate samples of 22.2 µM Aβ42 ADDLs and one sample containing both 22.2 µM Aβ42 and 5.55 µM PrP107–120 at a 4:1 molar ratio. For removing DMSO, dialysis was performed using Spectra/Por 3 dialysis kits  and 10 mM phosphate buffer, with a pH of 6.0, for 3 h at 4 °C. For the preparation of Aβ42 ADDLs + PrP107–120 sample, PrP107–120 was added at a final concentration of 5.55 µM after dialysis and was incubated for 2 h at 4 °C. Far-UV CD spectra were measured at 25 °C between 190–260 nm with a 1-nm spectral step size and 50-nm/min scanning rate on a JASCO J-810 spectropolarimeter  equipped with thermostated cell-holder attached to a Thermo Haake C25P water bath . A 1-mm thermostated quartz cuvette was used for all CD spectra. Spectra were blank-subtracted and normalized to mean residue ellipticity.To determine the PrPn-(2-hydroxyethyl)piperazine-n’-(2-ethanesulfonic) acid (HEPES) and NaHCO3, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 1% penicillin/streptomycin and maintained at 37 °C and 5% CO2. When the cells reached 90% confluence, they were split using 0.25% trypsin-EDTA to a maximum of 20 passages.Adherent human neuroblastoma SH-SY5Y cells  were cultured in Dulbecco’s Modified Eagle’s Medium , F-12 Ham with 25 mM 3 cells per well in a 96-well plate. After 24 h at 37 °C in a 5% CO2 atmosphere, cells were incubated for 24 h with different samples  DMSO and F-12 Ham medium, as described above). Final concentrations of Aβ42 and PrP107–120 in DMEM were 3 and 0.75 µM (m.e.), respectively, with a 4:1 molar ratio. Cells were incubated with 0.5 mg/mL of MTT solution in Roswell Park Memorial Institute (RPMI) medium for 3 h at 37 °C, then with lysis buffer  for 1 h at 37 °C. The optical density was measured at 590 nm by a microplate reader .SH-SY5Y cells were plated at a density of 15 × 103 cells per well. After 24 h, 600 µL of various samples  DMSO and F-12 Ham medium, as described above) were added to cells for 1 h at 37 °C. Final concentrations of Aβ42 and PrP107-120 in DMEM were 3 and 0.75 µM (m.e.), respectively. After washing with phosphate buffered saline (PBS), cells were loaded with a 4-µM Fluo-4 AM probe  for 10 min at 37 °C. Imaging was performed after excitation at 488 nm with a TCS SP8 confocal scanning microscopy system , using a Leica Plan Apo 63x oil immersion objective, taking a series of 1-µm-thick optical sections (1024 × 1024) through the cell depth for each sample and projecting them as a single composite image by superimposition. A minimum of four images were captured for each sample and four replicates were used for each condition. Images were analyzed using Image J software .SH-SY5Y cells were seeded on a glass coverslip in a six-well plate at a density of 40 × 103 cells per well. The medium was then replaced with 600 µL of various samples  DMSO and F-12 Ham medium, as described above) with final concentrations of 3 and 0.75 µM (m.e.) for Aβ42 and PrP107–120 in DMEM, respectively. After 45 min, 5 µM of 2,7-dichlorodihydrofluorecein diacetate probe  was added for 15 min at 37 °C. Finally, cells were washed twice in PBS and then fixed in 2% (w/v) paraformaldehyde for 10 min at room temperature. Cell image acquisition was performed using the TCS SP8 confocal system described in To detect intracellular accumulations of ROS, SH-SY5Y cells were cultured for 24 h on glass coverslips in a 6-well plate at a density of 40 × 103 cells per well, then exposed to 600 µL of various samples  DMSO and F-12 Ham medium, as described above) with final concentrations of 3 and 0.75 µM (m.e.) for Aβ42 and PrP107-120 in DMEM, respectively. After 1 h at 37 °C, cells were washed with PBS and stained with 1:1000 diluted Alexa Fluor 633-conjugated wheat germ agglutinin  for 15 min. Cells were rinsed again and fixed with 2% (w/v) paraformaldehyde for 10 min at room temperature. After cell membrane permeabilization with 0.5% BSA in PBS + 0.5% Triton X-100, cells were incubated with 1:800 diluted mouse monoclonal 6E10 antibody  for 1 h in 37 °C. After washing three times with PBS, cells were incubated with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse secondary antibody  for 1 h. All experiments were repeated three times and representative images of confocal microscopy are presented. Fluorescence was quantified with Image J software (NIH).SH-SY5Y cells were seeded in glass coverslips for 24 h in a 6-well plate at a density of 40 × 10107–120 on ADDLs structure, 2 µL of each sample  DMSO and F-12 Ham medium, as described above) were transferred onto a nitrocellulose membrane and allowed to dry for 15 min. For blocking the membrane, 1% (w/v) BSA in Tris-buffered saline and 0.1% Tween 20 (TBST) was used. After 1 h, the membrane was probed with 1:800 diluted mouse monoclonal antibody 6E10  or with 1:500 human antibody specific to ADDLs  at 4 °C overnight. The day after, the membrane was washed three times and subsequently incubated for 1 h at room temperature with 1:3000 peroxidase-conjugated anti-mouse secondary antibody  or 1:1000 peroxidase-conjugated anti-human secondary antibody . Imaging was performed using an Amersham imager 600 .To determine the effect of PrP42 ADDLs, Aβ42 + PrP107–120, Aβ42 ADDLs + PrP107–120 and PrP107–120, all dissolved in 2% (v/v) DMSO and F-12 Ham medium, as described above) or buffer as a blank. A 2- × 10-mm optical path-length was used and fluorescence spectra were recorded at 25 °C using the same PerkinElmer LS 55 fluorimeter described above, with excitation at 380 nm and emission at 400–650 nm.The ANS solution was prepared by dissolving 30 mg of 8-anilino-1-naphthalenesulfonic acid  in 10 mL of 25-µM phosphate buffer, with a pH of 6.0. After 20 min shaking, the solution was filtered by a 0.45-µm filter. ANS concentration was measured by optical absorbance at 375 nm and diluted in the same buffer to 55 µM. For each experiment, 450 µL of ANS solution was mixed with 50 µL of each sample  DMSO and F-12 Ham medium, as described above) with final concentrations of 3 and 0.75 µM (m.e.) for Aβ42 and labelled PrP107–120 in DMEM, respectively. After rinsing with PBS twice, 1:100 diluted fluorescent dye Hoechest  was added for 10 min at 37 °C. Following four washing steps with PBS, cells were fixed using 2% paraformaldehyde. Permeabilization of cells and Aβ42 staining were performed as described above. In another experiment, the concentration of labelled PrP107–120 was increased to 100 µM, and a 1:1 ratio for labelled PrP107–120 to Aβ42 was used in the initial pre-treatment in 2% (v/v) DMSO and F-12 Ham medium to prepare the Aβ42 + labelled PrP107–120 sample.BODIPY TMR-X NHS Ester  was dissolved in DMSO. PrP107-120 was labelled with BODIPY TMR-X NHS Ester, as described in 3 cells per well. After washing with PBS, cells were treated with 600 µL of labelled peptide at a final concentration of 0.75 µM for 1 h. After washing with PBS twice, 2% paraformaldehyde was added for 10 min. After fixation, cells were incubated with 1:250 diluted mouse monoclonal PrP (5B2) antibody  for 1 h at 37 °C. After washing with PBS, cells were incubated with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse secondary antibody . Imaging was performed after excitation at 488 and 561 nm to detect the cellular prion protein and the PrP107–120 peptide, respectively, using the TCS SP8 confocal system described in PrPp values < 0.05, < 0.01 and < 0.001, respectively.All data are presented as means ± S.E.M. (standard error of the mean). The difference between groups was analyzed using a Student’s t-test. The single (*/#), double (**/##) and triple (***/###) symbols refer to