Splitting water into hydrogen and oxygen with 3d transition metal molecular catalysts and light has been accomplished. 2 was employed as a low-cost scaffold with long-term stability for anchoring a phosphonic acid-modified nickel(ii) bis-diphosphine catalyst (NiP) for electrocatalytic proton reduction. A turnover number of 600 mol H2 per mol NiP was achieved after 8 h controlled-potential electrolysis at a modest overpotential of 250 mV. X-ray photoelectron, UV-vis and IR spectroscopies confirmed that the molecular structure of the Ni catalyst remains intact after prolonged hydrogen production, thereby reasserting the suitability of molecular catalysts in the development of effective, hydrogen-evolving materials. The relatively mild operating conditions of a pH 3 aqueous solution allowed this molecule-catalysed cathode to be combined with a molecular Fe(ii) catalyst-modified WO3 photoanode in a photoelectrochemical cell. Water splitting into H2 and O2 was achieved under solar light illumination with an applied bias of >0.6 V, which is below the thermodynamic potential (1.23 V) for water splitting and therefore allowed the storage of solar energy in the fuel H2.Splitting water into hydrogen and oxygen with molecular catalysts and light has been a long-established challenge. Approaches in homogeneous systems have been met with little success and the integration of molecular catalysts in photoelectrochemical cells is challenging due to inaccessibility and incompatibility of functional hybrid molecule/material electrodes with long-term stability in aqueous solution. Here, we present the first example of light-driven water splitting achieved with precious-metal-free molecular catalysts driving both oxygen and hydrogen evolution reactions. Mesoporous TiO The reliance on the long excited state lifetimes2 evolution.2 evolution with molecular catalysts driving the reduction and oxidation reactions in homogeneous solution has only been achieved in the context of oxidation of an organic substrate.2O into H2 and O2.The specific interest in molecular catalysts arises from the precise control afforded by modern synthetic chemistry over the individual catalytic centres, and therefore the opportunity to study the chemical and structural influences on catalysis.2 evolution catalyst limits the long-term applicability of the H2-evolving photocathode.A photoelectrochemical (PEC) approach to water splitting utilising immobilised molecular catalysts has many advantages. It allows efficient use of highly active and selective catalytic centres , can overcome kinetic restrictions from diffusion limitations, separates the redox half-reactions to avoid quenching mechanisms and allows separation of the gaseous products, as well as providing a platform for the systematic study of molecular catalysts under aqueous conditions without the requirement of water-solubility.ii) bis-diphosphine class of H2 evolution catalyst due to its high activity in both aqueous2 electrode. We have studied the activity and stability of this hybrid cathode, finding both to be excellent in mild aqueous conditions, which are essential for combination with a photoanode for solar water splitting. An Fe(ii)-based molecular catalyst immobilised on WO3 has shown a well-characterised increase in activity and selectivity for O2 evolution in mildly acidic aqueous conditions compared to the unmodified electrodes,2 hybrid cathode with a Fe-catalyst modified WO3 photoanode in a PEC water splitting cell.In this study, we present the first example of molecular catalyst-enabled water splitting using only Earth-abundant elements . We haveii) bis(diphosphine) complexes are a family of bio-inspired proton reduction catalysts,NiP  in excess of 700 mol H2 per mol NiP in photocatalytic schemes using a Ru(ii)-based dye and ascorbic acid as an electron donor, and has been demonstrated as a homogeneous electrocatalyst in pH 4.5 aqueous solution.27NiP features phosphonic acid groups, which are well-established for effective binding to TiO2 under mildly acidic aqueous conditions,2 facet.NiP make it an ideal candidate for immobilisation on metal oxide electrodes for single-site heterogeneous proton reduction.Ni catalysts immobilised onto mesoporous indium-doped tin oxide (ITO) electrodes.NiP displays substantially higher activity than the previously employed Co-based catalysts and we replaced the electrodegrading ITO cathode with robust TiO2. Although TiO2 is often considered as a classical insulator and therefore unsuitable as electrode material, it has previously been used for electrocatalytic proton reduction with immobilised H2-cycling enzymes known as hydrogenases,NiP for long-term reductive electrocatalysis.Attachment of molecular HNiP was synthesised and characterised as described previously,2 (mesoTiO2) electrodes were prepared by doctor blading a suspension of P25 TiO2  onto an FTO-coated glass substrates, followed by annealing at 450 °C. MesoTiO2 electrodes with a geometrical surface area of 1.0 cm2 were employed and scanning electron microscopy (SEM) revealed a film thickness of 4 μm  compound (0.5 mM) for 18 h at room temperature, followed by rinsing with methanol and drying under a stream of N2. The amount of NiP per geometric surface area of the TiO2 electrode was determined as 14.6 ± 2.0 nmol cm–2 by spectrophotometry (at λabs = 257 and 300 nm) following desorption of the catalyst from TiO2 with aqueous NaOH (0.1 M). The loading of NiP is in agreement with previous results, where a surface coverage of 53 nmol cm–2 was observed for a phosphonic acid-modified ruthenium complex on 10 μm thick mesoporous TiO2 electrodes.36m Fig. S1. ModificNiP-modified mesoTiO2 (TiO2|NiP) electrodes and bare mesoTiO2 in aqueous pH 3 electrolyte solution (0.1 M Na2SO4) are shown in 2 evolution activity of the immobilised Ni(ii) catalyst. The CVs of the unmodified TiO2 electrode show a typical ‘trumpet plot’ response as expected for a semiconductor electrode.Q = –0.6 mC at ν = 100 mV s–1), with an onset of approximately –0.1 V vs. the reversible hydrogen electrode (RHE), was followed by an oxidising current (Q = +0.4 mC) in the anodic scan. This observation was attributed to filling and emptying of the conduction band (CB) of TiO2. At pH 3, after modification with NiP, the oxidative current in the anodic scan is substantially diminished, with the charge ratio for the cathodic to anodic scans higher than 15 : 1, consistent with the consumption of the electrons from the CB of TiO2 for proton reduction catalysis.Cyclic voltammograms (CVs) of the resultant via the CB of TiO2 was obtained by spectroelectrochemistry.Eappl, of –0.43 V vs. RHE was applied to TiO2 electrodes, a blue colour was observed, and the corresponding increase in absorbance between 600 and 900 nm in the UV-vis spectrum is shown in Fig. S2a.3+, which is formed by filling the CB of TiO2.NiP, the increase in absorbance between 600 and 900 nm was still observed at Eappl = –0.43 V vs. RHE  current observed in the return scan of the CV in NiP on mesoTiO2, the absorbance at λ = 800 nm decayed to its original value within a few seconds (τ1/2 = 2.3 s) of the potential being removed, supporting the efficient release of CB electrons to NiP.Evidence that the electrons are transferred to the catalyst  Fig. S2b, but to otential . In the 2 production by the TiO2|NiP cathode was confirmed by controlled-potential electrolysis (CPE) in pH 3 aqueous electrolyte solution, and the amount of H2 produced alongside the theoretical maximum (assuming 100% faradaic efficiency) is shown in 2 electrodes modified with NiP were held at Eappl = –0.25 V vs. RHE in a two-compartment electrolytic cell for 8 h, during which time a charge of 2.02 ± 0.17 C passed. The current decayed slowly during the first few hours, which is consistent with the good stability of the molecular catalyst in aqueous solution .Sustained electrocatalytic H Fig. S3a.20,27 AnNiP was desorbed from mesoTiO2 with NaOH (0.1 M) after 8 h CPE and the UV-vis spectrum of the resultant solution matched a reference spectrum recorded from freshly-modified electrodes treated in the same way, suggesting minimal degradation of the ligand framework of NiP  spectroscopy. Fig. S4bNiP powder, TiO2|NiP before and after 4 h CPE at –0.25 V vs. RHE, and TiO2 (treated with Na2SO4 electrolyte solution). Stretches at 1260 cm–1  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O), 1440 and 1510 cm–1 (NiP ligand) were present in the reference NiP spectrum and TiO2|NiP before and after CPE, but not the TiO2 background, reasserting the molecular integrity of the catalyst surviving many turnovers on the electrode surface.Further evidence for the retention of molecular 2 displayed substantially improved performance to mesoITO, which has previously been used as a substrate for a phosphonate-bearing Co-based catalyst.NiP loading per geometric surface area of 25 nmol cm–2. Although the CV supports a catalytic current for ITO|NiP in an aqueous pH 3 electrolyte solution .We have also tested the influence of Oy Fig. S7, achievi2 as an inexpensive, easily-prepared mesoporous cathode material for immobilisation of phosphonate-bearing molecular synthetic catalysts, particularly in terms of the stability of both the material itself and the interaction between the TiO2 and the molecular catalyst, enabling high turnover numbers to be reached and substantial amounts of H2 being generated at a modest overpotential. Our approach thereby complements previous work, where Ni(ii) bis(diphosphine) complexes were attached onto electrode surfaces such as silicon2|NiP is easier to assemble, requires a less expensive substrate, and can be studied spectroelectrochemically. Furthermore, the transparent nature of TiO2 to visible light and the demonstrated high activity in mild aqueous conditions make TiO2|NiP a suitable cathode material for combination with a photoanode to allow for full water splitting, which is difficult to achieve for cathodes operating under the less sustainable conditions used previously.2 as cathode material in this work is also of interest in the context of current work on its use as a protection layer for photocathode materials such as Cu2O,2/CdS,2 production in the presence of a Pt catalyst. Employment of phosphonated molecular catalysts such as NiP on such TiO2 layers appears as a promising approach to replace expensive noble metals.These results establish mesoTiO2 sensitised with molecular dyes, including those based on Ru(ii),ii)3 is notable for good stability under the mildly acidic aqueous conditions required for operation with TiO2|NiP,2O3 and BiVO4 are typically studied in alkaline2 evolution of WO3 in aqueous pH 3 Na2SO4 solution.3,ii) catalyst based on a phosphonic acid-modified tris(2-picolyl)amine (TPA) ligand, the unmodified triflate-coordinated analogue of which is known to perform water oxidation homogeneously at low pH in the presence of a chemical oxidant.12Photoanodes composed of molecular hybrid materials developed thus far largely fall into the category of n-type TiOvia a multi-step chemical synthesis shown in 1. The phosphonic acid was introduced first by Pd-catalysed cross coupling2 and subsequently hydrolysed in aqueous HCl to give TPAp1·3HCl. The phosphonic acid-modified TPA ligand was coordinated to FeCl2 in methanol in the presence of Et3N to precipitate FeP . Immobilisation was achieved by submersion in an FeP solution (2 mM in methanol) overnight at room temperature, and representative CVs are shown in Fig. S9.FeP electrodes displayed a reversible redox wave in pH 3 aqueous solution at E1/2 = 0.7 V vs. RHE and a linear dependence of the peak current with scan rate supports the immobilisation of FeP on the metal oxide electrode surface.The electrochemical response of immobilised 3 nanosheet (nanoWO3) electrodes were synthesised hydrothermally onto a WO3 seed layer deposited on FTO as previously described  overnight at room temperature in the dark, and the UV/vis spectrum shows a small increase in absorbance between 400 and 450 nm  from FeP-modified WO3 films in comparison to unmodified films and those treated with FeCl2 and the TPAp1 ligand was demonstrated by linear sweep voltammetry with chopped illumination . This result suggests an improved photoelectrocatalytic water oxidation by the WO3 film modified with the molecular catalyst, but not the constituent metal salt or ligand in isolation. This result was corroborated by CPE at Eappl = 1.0 V vs. RHE under illumination, with the charge equivalents and amount of O2 produced shown in 3|FeP was found to produce 3.7 ± 0.4 μmol O2 with 40 ± 4% faradaic efficiency, compared to 1.8 ± 0.3 μmol O2 with 21 ± 2.1% for the unmodified electrode. The low efficiencies can be explained by competing electrolyte oxidation and incomplete oxidation of water known to occur under acidic aqueous conditions,ii) catalyst matched the precedent set previously.23WOFig. S10a, consistFig. S10b. Fig. 3a3|FeP was found to vary with the wavelength of incident monochromatic light in accordance with the UV/vis spectrum of WO3  of WOFig. S10a, and mad3|FeP photoanode displays good water oxidation activity and is compatible with the H2-evolving TiO2|NiP cathode in an aqueous pH 3 electrolyte solution. 2|NiP cathode and WO3|FeP photoanode are combined in a two-electrode PEC configuration, the onset of photocurrent should be at an applied voltage of approximately 0.6 V. This is lower than the thermodynamic potential requirement for water splitting (1.23 V) and the proposed device should have capacity for solar energy storage in the fuel H2.The WO3|FeP as the photoanode under periodic simulated solar light irradiation and TiO2|NiP (shielded from the illumination to avoid UV band gap excitation of TiO2) as the cathode, with the electrodes separated by a proton-permeable Nafion membrane to prevent gas diffusion between the cathodic and anodic compartments  photon-to-current conversion efficiency  taken after 45 s illumination at each Uappl under full simulated solar spectrum irradiation with a light intensity (P) of 100 mW cm–2.We therefore assembled a two-electrode PEC cell, with WOartments . As predthe TiO2 . The appcy ABPE,  for the Uappl = 1.0 V with an ABPE = 0.035%. The low value for ABPE is due to incomplete use of the solar spectrum by WO3 shown in Fig. S10a2 production, unlike the APBE presented here.19The ABPE was found to have a maximum at 2 and H2 are summarised in 2 were detected by a fluorescence sensor  for 1 h, with the results summarised in 2  and O2  evolution activity than at Uappl = 1.1 V. These results demonstrate that the molecular catalysts based on Earth-abundant transition metals are also capable of driving water splitting at a higher rate if better electrode materials become available. A tandem PEC device with a suitable photocathode modified with NiP wired to WO3|FeP would dramatically reduce the applied bias and result in higher photocurrents and ABPE in the future.Without FeP are known to remain molecular during water oxidation in homogeneous aqueous acidic conditions, but to oxidise to catalytically-active iron oxide nanoparticles under basic conditions.NiP in solution or in suspensions with semiconducting nanoparticles.NiP-modified mesoTiO2 electrode. Before electrolysis, the electrodes were purple in colour and showed a broad band at λmax = 520 nm in the UV-vis spectrum of NiP. The purple colour with the band at λmax = 520 nm were qualitatively retained following three-electrode CPE for 2 h and two-electrode solar water electrolysis coupled to WO3|FeP for 1 h, indicating that molecular NiP remained on mesoTiO2.The study of molecular catalysts under strongly reductive and oxidative conditions requires an assessment of their integrity during catalysis.2|NiP and 1 : 4 : 1 for WO3|FeP, before two-electrode PEC water splitting were as expected  catalyst slowly desorbs from the electrode surface during photoelectrocatalysis,FeP. Therefore, the limiting electrode in this PEC water splitting cell is the FeP-modified WO3 photoanode.X-ray photoelectron spectroscopy (XPS) on both modified electrodes before and after running 1 h PEC water splitting at an applied bias of 1.1 V were performed to analyse the possible formation of metal-containing deposits. The Ni(2p) or Fe(2p) : N(1s) : P(2p) ratios, of approximately 1 : 4 : 8 for TiOexpected . In agre Fig. S12, suggest2|NiP. NiP remains intact during electrolysis immobilised on mesoTiO2 electrodes. This result emphasises the suitability of molecular electrocatalyst design even when working on heterogeneous catalytic systems.Peaks corresponding to Ni(2p), N(1s) and P(2p) were retained after catalysis  and S13†ii) bis(diphosphine) molecular catalyst and mesostructured TiO2, which is normally considered an insulator in the absence of UV irradiation and therefore of limited use in electrocatalysis. We observed, from complementary cyclic voltammetry and spectroelectrochemistry, that at mildly reducing potentials electrons are transferred first to the CB of TiO2, and then to NiP for proton reduction catalysis. During long term controlled-potential electrolysis, sustained H2 production was observed at an applied potential of Eappl = –0.25 V vs. RHE, with a NiP-based turnover number of 600 achieved after 8 hours. Characterisation and quantification of the catalyst after electrolysis revealed not only that the molecular structure was intact, but also 75% of the NiP initially present remained on the electrode surface. The modified TiO2 performed considerably better than an analogous mesoporous ITO electrode, which underwent degradation under reducing conditions. Therefore, the TiO2|NiP cathode exhibited excellent stability in terms of the material itself, the molecular structure of the catalyst, and the attachment between the two in mildly acidic aqueous conditions. These results establish TiO2|NiP as not only an effective H2 evolving electrode in its own right, but also suggest potential as a catalytically-active protection layer for photocathodes due to its ease of preparation, transparency to visible light, stability and high activity at a modest overpotential.We have presented a hybrid proton reduction cathode based on a phosphonated Ni from Evonik Industries. NiP2 prior to use, or in the case of methanol purchased as dry solvent and stored over molecular sieves. Solvent mixtures are reported as vol : vol ratios. Standard Schlenk line techniques were used where required. 1H, 13C and 31P NMR measurements were performed on a Bruker DPX400 spectrometer. Mass spectrometry measurements were performed on a Waters Micromass Quattro LS ESI or ThermoScientific Orbitrap Classic instrument . SEM was performed on a FEI Phillips XL30 field emission gun SEM instrument. XPS was performed by the Nexus facility at the University of Newcastle on a K-Alpha  spectrometer utilising a monochromatic Al-Kα X-ray source . Survey spectra were collected with a pass energy of 200 eV and 3 sweeps, while high resolution spectra were collected at a pass energy of 40 eV with 10 sweeps. Elemental analysis was carried out by the University of Cambridge Microanalysis Service using a Perkin-Elmer 240 Elemental Analyser. UV-vis absorption spectroscopy was performed using a Varian Cary 50 spectrophotometer. Where stated, a diffuse reflectance accessory for the spectrophotometer was used, with a Spectralon reference as a background. UV-vis spectra (reflectance mode) of WO3 were recorded on an Edinburgh Instruments FS5 spectrofluorometer equipped with an integrating sphere by running a synchronous scan (λex = λem) and subtracting a scan of the sphere background. ATR-FTIR measurements were performed on a Nicolet iS50 FTIR spectrometer.All starting materials were obtained from commercial sources and used without further purification, unless otherwise stated. ITO nanopowder (<50 nm particle size) was obtained from Sigma-Aldrich and P25 TiOin vacuo. Dry CH2Cl2 (50 mL) was added, followed by 4-bromo-2-pyridinecarboxaldehyde  and di-(2-picolylamine) . The mixture was degassed by three freeze–pump–thaw cycles, purged with N2 and stirred under N2 at room temperature for 2 d. Saturated aqueous NaHCO3 (20 mL) was added, and the biphasic mixture stirred for 1 h. The crude product was extracted in CH2Cl2 (3 × 25 mL), washed with brine (20 mL), dried over MgSO4 and the solvent removed in vacuo. Purification was achieved by flash column chromatography on silica (pre-deactivated with Et3N), eluting in CH2Cl2 : Et3N (98 : 2) to yield 1 as a pale yellow oil . HR-MS calc. for [C18H18BrN4]+: 369.0709, found: 369.0724 (100% peak); 1H-NMR  δ/ppm: 8.58 , 8.36 , 7.80 , 7.70 , 7.56 , 7.34 , 7.18 , 3.93 , 3.91 ; 13C NMR  δ/ppm 149.82, 148.46, 137.52, 136.93, 133.62, 126.67, 125.82, 123.75, 122.74, 120.88, 60.40, 59.33.This compound was synthesised by adaptation of a literature procedure for tris(2-pyridylmethyl)amine.ii) acetate  and 1,1′-bis(diphenylphosphino)ferrocene  were dried in vacuo. In a separate pre-dried flask, 1  was added and the flask evacuated and purged with N2 three times. The compound was dissolved in dry acetonitrile (6 mL) and added to the reaction flask under N2. Diethyl phosphite  and Et3N (1 mL) were added and the mixture was degassed by three freeze–pump–thaw cycles. The flask was heated to 70 °C under N2, sealed, and stirred at 70 °C for 2 d. The reaction mixture was cooled, the solvent removed in vacuo, and the product purified by flash column chromatography on silica (pre-deactivated with Et3N), eluting with CH2Cl2 : Et3N : CH3OH (97.5 : 2 : 0.5) to yield 2 as a yellow oil that discoloured quickly and solidified on standing . HR-MS calc. for [C22H28N4O3P]+: 427.1894, found 427.1875 (100% peak); 1H-NMR  δ/ppm: 8.68 , 8.53 , 7.96 , 7.67 , 7.58 , 7.50 , 7.15 , 4.06–4.24 , 3.97 , 3.90 , 1.34 ; 13C  δ/ppm: 160.74 , 159.45 (s), 149.78 , 149.48 (s), 138.16 , 136.86 (s), 124.92 , 123.83 , 123.39 (s), 122.47 (s), 63.07 , 60.60 (s), 60.37 (s), 16.72  31P-NMR  δ/ppm: –16.31; IR ν = 1250 cm–1  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O).In a pre-dried Schlenk flask with a Teflon screw-cap, palladium was dissolved in 18% aqueous HCl (4 mL) and refluxed for 18 h. The reaction mixture was cooled to r.t. and the solvent was removed in vacuo and the brown oily crude dissolved in CH3OH. Precipitation with EtOAc followed by filtration and washing with EtOAc yielded TPAp1·3HCl as a hygroscopic off-white powder . MS calc. for [C18H20N4O3P]+: 371.12 (100.0%), found: 371.0 (100%); 1H-NMR  δ/ppm: 8.65 , 8.60 , 8.42 , 7.99 , 7.86 , 4.35 , 4.27 ; 13C-NMR  δ/ppm: 151.42 , 151.00 (s), 147.52 (s), 144.34 , 142.09 , 141.91 (s), 127.85 , 127.53 (s), 127.16 , 126.78 (s), 56.37 (s); 31P-NMR  δ/ppm: –4.36; IR ν = 1170 cm–1  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O); EA calc. for C18H26Cl3N4O5P (TPAp1·3HCl·2H2O) C, 41.92; H, 5.08; N, 10.86; P, 6.01, found C 42.51, H 5.01, N 10.42, P 5.81.Compound TPAp1·3HCl  was dissolved in dry CH3OH under N2. Dry Et3N  was added, and the solution stirred under for 30 min. This solution was added to a degassed solution of anhydrous FeCl2  in CH3OH (2 mL). The solution was freeze–pump–thaw degassed three times, and stirred under N2 for 2 h. The solvents were removed in vacuo, CH3CN (4 mL) was added, and the suspension stirred under N2 for 12 h to dissolve the Et3NHCl. The suspension was filtered off and the precipitate dried in vacuo to give FeP as a red powder . MS calc. for [Fe(TPAp1)Cl]+: m/z: 461.022 (100.0%), found 460.97 (100%); EA calc. for C19H23ClFeN4O3P [TPAp1FeCl(OMe)] C, 46.32; H, 4.50; N, 11.37; found C, 46.02; H, 4.85 N, 11.60; IR ν = 1170 cm–1  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O).In a pre-dried Schlenk flask, 2 of dimensions 3.0 cm × 1.0 cm were cleaned by heating at 70 °C in a 5 : 1 : 1 solution of H2O : H2O2 (30% aq.) : NH4OH (conc. aq.) for 30 min, followed by rinsing with H2O and drying at 180 °C for 1 h. Suspensions of ITO (20% by weight of ITO in a 5 M acetic acid solution in ethanol) and TiO2 nanoparticles (100 mg TiO2 and 50 mg poly(ethylene glycol) in approximately 1 mL ethanol) were applied to the transparent conducting oxide-coated glass slides using the doctor blading method using a Scotch tape mask with aperture dimensions of either a 6 mm diameter circle (for cyclic voltammetry) or a 0.7 cm × 1.5 cm rectangle (for (photo)electrolysis). The slides were then annealed at 450 °C for 0.5 h (mesoTiO2) or at 400 °C for 1 h (mesoITO).Prior to preparation of mesoporous electrodes, glass slides coated with indium-doped tin oxide (ITO) for mesoITO or fluorine-doped tin oxide (FTO) for mesoTiO2 and mesoITO electrodes were cleaned and dried with ammonia/hydrogen peroxide (see above) and/or using a BioForce UV/ozone cleaner. Immobilisation was achieved by submersion of the electrodes in a 0.5 mM solution of NiP in methanol for 18 h. The coverage of NiP was quantified by submersion of the modified electrodes in a NaOH(aq) solution (0.1 M) for 30 min, followed by UV-vis spectroscopy of the resultant solution and comparison with calibration data at λ = 257 and 300 nm.The mesoTiO3 electrodes were prepared as previously described by hydrothermal synthesis,2 for three-electrode measurements and 0.5 cm2 for two-electrode measurements.WO3+ potentiostats. All electrochemical measurements were performed in a Na2SO4 (0.1 M) aqueous solution, adjusted to the desired pH by titration with H2SO4 at room temperature and purged with N2 unless otherwise stated. All electrochemical experiments on the individual TiO2|NiP cathode or WO3|FeP photoanode were performed in a three-electrode configuration with a Pt counter electrode and a Ag/AgCl/KCl(sat) reference electrode, whereas full water splitting CPE experiments were performed in a two-electrode setup with TiO2|NiP directly (shielded from illumination) connected to WO3|FeP in a two-compartment airtight cell. Irradiation was provided through a quartz illumination window and the cathode was separated from the anode by a Nafion membrane. Simulated solar irradiation was provided by a Newport solar light simulator with an AM1.5G filter and a water IR filter. TiO2 was shielded from light to avoid UV band gap excitation. Light intensity was measured using an International Light Technologies 1400 photometer. IPCE measurements were performed using monochromatic light provided by a LOT 300 W Xe lamp equipped with a MSH300 monochromator (FWHM = 5 nm). IPCE was calculated using eqn (2).Iel is the electron flux of the external circuit (mol m–2 s–1), Iph is the incident photon flux (mol m–2 s–1), j is the measured photocurrent density (μA cm–2), F is the Faraday constant (96 484 A s mol–1), λ is the wavelength of light (nm), W is the incident power of the monochromated light (W m–2), NA is Avogadro's number (6.022 × 1023 mol–1), h is Planck's constant (6.626 × 10–34 J s) and c is the speed of light (2.998 × 108 m s–1).All electrochemical measurements were performed on Ivium Technologies CompactStat or IviumStat, or PalmSens EmStat or MultiEmStatResistance between electrodes in the two compartments was found to be <100 Ω by electrochemical impedance spectroscopy and the reported voltages are not corrected for cell resistance. The experiments were performed at room temperature, and the cell was purged with an inert gas before each experiment. All reduction potentials from the three-electrode experiments are reported against RHE using eqn (3).2 in the beampath of a Varian Cary 50 spectrophotometer.Spectroelectrochemical measurements were performed in a borosilicate glass one-compartment electrochemical cell purged with N2 regime using a mass-flow controller. The chamber containing the oxygen probe  was degassed by purging the solution with N2 for 30 min. Background O2 was then recorded for 30 min, before commencement of the photoelectrolysis experiment. Finally, a further 30 min background was run after ceasing illumination and the background levels of O2 were subtracted. The total amount of O2 produced was obtained using the ideal gas law for O2 in the headspace, and Henry's law for dissolved O2.Oxygen detection was performed using an Ocean Optics NeoFox phase fluorimeter equipped with a FOSPOR probe calibrated in the 0–3% O2 quantification, the cell was purged with N2/CH4 (2%) for 10 min prior electrochemical experiments. The H2 produced was quantified by an Agilent 7890A gas chromatograph, with CH4 as an internal standard. For experiments performed under air, CH4 was used as an external standard. Gas detection experiments were repeated 3 times and values given as an average of the three ± standard deviation.For HSupplementary informationClick here for additional data file.


Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are promising candidates to treat myocardial infarction and other cardiac diseases. Such treatments require pure cardiomyocytes (CMs) in large quantities.Methods: In the present study we describe an improved protocol for production of hiPSC-CMs in which hiPSCs are first converted into mesodermal cells by stimulation of wingless (Wnt) signaling using CHIR99021, which are then further differentiated into CM progenitors by simultaneous inhibition of porcupine and tankyrase pathways using IWP2 and XAV939 under continuous supplementation of ascorbate during the entire differentiation procedure.Results: The protocol resulted in reproducible generation of >90% cardiac troponin T (TNNT2)-positive cells containing highly organized sarcomeres. In 2D monolayer cultures CM yields amounted to 0.5 million cells per cm2 growth area, and on average 72 million cells per 100 mL bioreactor suspension culture without continuous perfusion. The differentiation efficiency was hardly affected by the initial seeding density of undifferentiated hiPSCs. Furthermore, batch-to-batch variations were reduced by combinatorial use of ascorbate, IWP2, and XAV939.Conclusion: Combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling and continuous ascorbate supplementation, enable robust and efficient production of hiPSC-CMs. The pumping function of the heart is of vital importance to generate sufficient blood flow and pressure. Different pathologies impair cardiac function; cardiac infarction results in an acute loss of large quantities of contractile cells and fibrotic remodeling of the tissue. As a result heart failure can occur. Adult cardiomyocytes (CMs) do not divide and therefore novel approaches need to be identified to overcome the minor regenerative capacity of the heart, provide new CMs to the diseased organ and reduce the size of the scar and hence the risk of heart failure. Today, neither pharmacological nor advanced surgical interventions are capable of restoring the lost contractile function of the infarcted area in vitro and generate teratomas in vivoHuman iPSCs have been discovered in 2007 In vivo physiological pathways are adapted for differentiating CMs at early stage in vitro and biological morphogens are replaced by synthetic small molecules that can be produced at comparable low costs https://www.wicell.org/). Detailed information about the NP0040-8 and NP0141-31B cell lines can be obtained from the online hPSC line registry hPSCreg (https://hpscreg.eu/) under names UKKi011-A and UKKi032-C, respectively. hiPSC lines were cultured in E8 medium composed of DMEM / F12 (1:1) + Glutamax  supplemented with 64 μg/mL L-ascorbic acid phosphate magnesium n-hydrate , 20 μg/mL insulin , 5 μg/mL transferrin , 14 ng/mL sodium selenite , 100 ng/mL heparin sodium salt , 100 ng/mL fibroblast growth factor 2 , and 2 ng/mL transforming growth factor β  2 Matrigel Matrix  for one hour at room temperature, and incubated in a humidified incubator at 37°C, with 5% CO2. Cells were passaged every 4 days at 1:20 splitting ratio using 0.5 mM EDTA . Following dissociation of hiPSCs E8 medium was supplemented by 5 μM of Rho Kinase (ROCK) inhibitor  for the first two days after passage. Medium was changed every other day, and 0.5 mL penicillin and streptomycin  per mL culture medium were supplemented in both hiPSCs and hiPSC-CM culture media.Human iPSC lines used in this study were the NP0040-8  and NP0141-31B  cell lines that were generated by Saric group, and the IMR90 cell line that was kindly provided by WiCell  with 1× B27 supplement without insulin  and 8 μM CHIR99021  for 24 hours.  After 24 hours, culture medium was changed into RPMI 1640 with 1× B27 supplement without insulin for 48 hours. 72 hours post CHIR99021 supplementation the culture medium was changed into RPMI 1640 with 1× B27 supplement without insulin and 5µM IWP2  for 48 hours. On day 5, 7, and every three other days medium was changed in RPMI 1640 with 1× B27 supplement without (till day 7) and with insulin (from day 7 onwards), the cardiomyocyte contraction was observed on day 12.A state-of-the-art differentiation protocol for hiPSC-CMs using small molecules to induce differentiation was adapted from Lian and colleagues 2. The incubation time for dissociation of hiPSCs was 3 minutes to obtain small clusters and 7 minutes for single cells. Either, 1:20 splitting ratio of small clusters, or 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3 and 0.4×106 cells/cm2 were seeded on Matrigel-coated six-well plates and maintained in 2 mL of E8 medium for four days. For the first two days the culture medium was supplemented with 5 μM ROCK inhibitor. After four days the culture medium was changed to RPMI 1640 with 1× B27 supplement without insulin , 50 μg/mL ascorbate and 8 μM CHIR99021. This day was defined as day 0 of differentiation. Exactly after 24 hours, the medium was completely changed to RPMI 1640 with 1X B27 supplement without insulin, and 50 μg / mL ascorbate. At day 3, 72 hours after initiation of differentiation half of the medium was changed to RPMI 1640 with 1× B27 supplement without insulin, 50 μg / mL ascorbate, and final concentrations of 5 μM IWP2 , and 5 μM XAV939  for 48 hours. From day 5 onwards the culture medium was changed to RPMI 1640 plus 1× B27 supplement without insulin and with 50 μg/mL ascorbate and replaced every 3 days.Our optimized protocol started from washing hiPSCs culture with Dulbecco's phosphate buffered saline without calcium and magnesium (DPBS-/-), and 1 mL of 0.5 mM EDTA (room temperature) was added into each well of a six-well plate and incubated in a humidified incubator at 37°C, with 5% COBioreactor preparation: Bioreactor  inner vessel walls were siliconized with 1 mL Sigmacote . Vessels were gently swirled to cover the whole surface with Sigmacote. The remaining Sigmacote was collected and reused. Coated vessels were left under the fume hood for one hour to remove the residual amount of liquid Sigmacote.2, 5% CO2 at 37°C and three standard liters per hour (sL / h) overlaying flow gas. After differentiation of CMs, the bioreactor vessels were washed with distilled water and 70% ethanol.To prepare the bioreactor system  a calibration of the pH sensor was performed at pH 4 and 7. All bioreactor parts were assembled including: glass vessel, dissolved oxygen (DO) sensor, pH sensor, and head caps. The vessel was autoclaved at 121°C for 15 minutes, and cooled to room temperature. Under a laminar flow hood 100 mL of E8 medium were added to each of the 250 mL volume capacity glass vessel . The vessel was placed back onto the temperature controller. Overnight polarization and calibration of DO sensor were performed at 60 revolutions per minute (rpm) agitation with 21% OhiPSC-CM differentiation: hiPSCs were cultured on Matrigel-coated 100 mm dishes  and incubated in a humidified incubator at 37°C, with 5% CO2. hiPSCs were cultured in E8 medium for about one week with daily medium change until 85% cell confluency was reached. Cells were washed with DPBS-/-. For dissociation, cells were incubated with 1-2 mL 0.5 mM EDTA per plate for 7 minutes at room temperature, the dissociated cells were passed through a 40 μm filter  and collected in a 50 mL Falcon tube with 10 mL E8 medium containing 10 μM ROCK inhibitor. Cells were counted by an automatic cell counter . 40 million single cells were re-suspended in 25 mL E8 medium supplemented with 10 μM of ROCK inhibitor and this suspension was inoculated into the bioreactor vessel containing 100 ml E8 medium containing 10 μM of ROCK inhibitor in order to obtain a final volume of 125 mL per vessel. Cultures were agitated at a propeller speed of 60 rpm, passed with 21% O2 and 5% CO2 by 3 sL/h overlay gassing and maintained at 37°C. After 48 hours of incubation (day -4 and -3) in E8 medium supplemented with ROCK inhibitor, the medium was changed to E8 medium without ROCK inhibitor and hiPSC clusters were incubated for additional 48 hours (day -2 and -1) until initiation of differentiation.At day 0, the cardiac differentiation was induced by a complete change of the medium to RPMI 1640 supplemented with 1× B27 without insulin, 50 μg/mL ascorbate and 8 μM CHIR99021. After 24 hours, the complete medium was changed into RPMI 1640 containing 1× B27 without insulin and 50 μg/mL ascorbate, and cells were incubated for another 48 hours. At day 3, half of the medium was removed and replaced with fresh RPMI 1640 supplemented with 1× B27 without insulin, 100 μg/mL ascorbate, 10 μM IWP2 and 10 μM XAV939 for 48 hours. From day 7 of differentiation onwards, the medium was replaced every three days with fresh RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate. At day 18 of differentiation, CM clusters were collected in a 50 mL Falcon tube and allowed to settle on the bottom of a tube by gravity. Clusters were then washed with DPBS-/- and dissociated by using 0.05% Trypsin-EDTA . The dissociated cells were passed through an EASYstrainer filter with 40 μm pore size and single cells were counted by an automatic cell counter.6) were plated on Matrigel-coated round cover slips in 12-well plates and incubated for two days in a humidified incubator at 37°C and CO2 level of 5% in 1 mL/well RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate. Alternatively, immunocytochemistry was performed directly with hiPSC-CMs that were generated on Matrigel-coated square cover slips in six well plates. The wells were washed with 1× DPBS-/-, fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, and permeabilized with 0.5% Triton X-100 in DPBS-/- for 15 minutes. Intact cardiac clusters were fixed and permeabilized with 100% methanol for 15 minutes. Unspecific antibody binding sites were blocked by incubation in 1 mL per well  of 3% bovine serum albumin (BSA)  in DPBS-/- for one hour at room temperature. For preparation of cryoslices, cardiac clusters were embedded in Tissue-Tek  and stored frozen at -80°C, and cryo-sliced at 8 µm thickness. Cells or cardiac cluster samples were incubated overnight at 4°C in 3% BSA containing 1:200 dilution of troponin T (TNNT2) rabbit polyclonal IgG  and 1:200 dilution of α-actinin (ACTN2) mouse monoclonal IgG1 . The next day, cells were washed three times with DBPS-/- and incubated for 60 minutes at room temperature in the dark and 3% BSA containing Hoechst 33342 and Alexa Fluor 555 goat α-rabbit IgG  and Alexa Fluor 647 goat α-mouse IgG , both at a concentration of 1 µg/ml. Following the final washing steps, cells were mounted with 5 µL SlowFadeTM Diamond Antifade Mountant  and examined by confocal fluorescent microscopy .Dissociated single cells  and left for 20 minutes at 4°C for further fixation and permeabilization. Cells were centrifuged at 120×g and washed by 2 mL DPBS-/- to remove the residue of methanol and then the pellet was incubated for one hour, or overnight in 100 µL of blocking buffer (0.5% BSA and 0.1% Triton X-100 in DPBS-/-) containing 1:50 dilution of troponin T (TNNT2) mouse monoclonal IgG2a  or normal mouse monoclonal IgG2a , while for double staining Anti-Cardiac Troponin T-FITC  and Anti-α-Actinin (Sarcomeric)-PE , or Anti-Myosin regulatory light chain 2 atrial isoform conjugated with allophycocyanin (Anti-MLC2a-APC)  and Anti-Myosin regulatory light chain 2 ventricular isoform conjugated with phycoerythrin (Anti-MLC2v-PE)  were used. After washing in blocking buffer the cells were incubated for 30 minutes in 100 µL of blocking buffer containing 1 µg/mL secondary antibody Alexa Fluor 555 goat anti-mouse IgG (H+L)  in the dark and at room temperature. Cells were washed two to three times with 2 mL of DPBS-/-, the final cell pellet was re-suspended in 250 μL DPBS-/- and cells analyzed by flow cytometry . Data was evaluated using FCS express 6 .1.5×106 hiPSC-CMs were added into RNase free 1.5 tubes, washed by PBS (-/-), and centrifuged at 300 g for 2 minutes at 4 °C. The pellet was re-suspended in 1 mL TRIzol , repetitive pipetting and mixing on a vortex mixer were performed to lyse CMs for 30 - 60 seconds. Lysate was incubated for 5 minutes at RT. For phase separation of RNA from phenol 0.2 mL chloroform  per tube were added and carefully mixed by hand and centrifuged at 12000g for 15 minutes at 4 °C. 350 μL of colorless supernatant were transferred into new RNase free 1.5 mL tubes and 350 µL 100% ethanol  were added, and vortexed well. 700 μL of mixed sample were passed through a spin cartridge with collection tube (provided with the kit), and centrifuged at 12000 g for 1 minute at RT. The spin cartridge was washed by 500 µL wash buffer II  and centrifuged at 12000 g for 15 second at RT. 22 μL RNase free water were used to elute RNA into 1.5 RNase free tubes, RNA aliquots were stored at -80 °C.Total RNA from hiPSC-CMs was isolated by PureLink RNA Mini Kit . Briefly, 1 x 10For transcriptome analysis 5.5 µg fragmented biotin-labeled ds cDNA from cardiomyocytes were hybridized to Clariom™ S arrays . After staining, arrays were scanned with Affymetrix Gene-Chip Scanner-3000-7G while quality control analysis was performed using Affymetrix GCOS software. Transcriptome analysis was done at the transcriptomics core facility at the Center for Molecular Medicine Cologne (CMMC).6 hiPSC-CMs were seeded on 10 µg / cm2 glass bottom dishes  pre-coated with Matrigel and incubated until day 20 - 40 in a humidified incubator at 37°C and CO2 level of 5%. Per well 1 mL RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate was added, complete medium change was performed every other day. At day 20, 30, and 40 RPMI 1640 was discarded and hiPSC-CMs were incubated in HEPES-buffered Tyrode´s solution supplemented with sodium pyruvate. Cells were loaded for 30 minutes at 22 - 23 °C with 5 μM Fluo-4 AM calcium indicator , and measurements were conducted using an Olympus FluoView1000 confocal system. To examine the influence of adrenergic stimulation on frequency and kinetics of calcium transients, confocal scans were performed before and after addition of isoproterenol  to a concentration of 10µM.At day 18 of CM differentiation 1.2 X 106 hiPSC-CMs were seeded on 10 µg / cm2 Matrigel pre-coated “E-plate Cardio 96” multiwell plates containing gold electrode arrays fused to the bottom of the plates (ACEA Biosciences) and incubated for further 6 - 7 days in 0.150 mL / well RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate. The E-plate was transferred into an xCELLigence Real Time Cell Analyser Cardio (RTCA) instrument  to monitor hiPSC-CMs contracting activity for 4 - 5 days. The measurement is based on fluctuations of the impedance of the cell layer when CMs contract in a syncytium. Once the syncytium monolayer hiPSC-CMs contractions were observed, the hiPSC-CMs were exposed to 10 µM Iso, and 10 µM carbachol (CCH) in two different row of E-plate for two days.The hiPSC-CMs beating rate was monitored for totally 9 days (2 days with drugs) and analyzed using the RTCA Cardio software version 1.0 (ACEA Biosciences).hiPSC-CMs generated in 2D and 3D culture were dissociated at day 18 of differentiation. 0.15 X 106 / cm2, and cultured inside 12-well plates in 2 mL RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate at 37 °C and 5% CO2. After 3 (n=3) or 11 days (n=4), the cover slips with hiPSC-CM were incubated for 15 min with a membrane potential-sensitive fluorescent dye (1uM DI-4-ANEPPS) and Blebbistatin (10 uM) to remove motion artefacts. After that the coverslips were placed in the optical mapping setup and superfused with Tyrode's solution (36±0.2˚C) containing (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, and HEPES 5.0 (pH7.38). The monolayers were stimulated at 2 beat per minute at twice diastolic threshold with a pulse width of 2 ms using an extra cellular electrode. Optical action potentials were recorded using a CMOS camera  and analyzed using custom-made software hiPSC-CMs were seeded on Matrigel-coated round cover slips (diameter of 2 cm) at a density of 1.7-2.3 x 10GraphPad Prism software version 5 was used for statistical analysis and graph drawing. Where stated, graphs were created using the Olympus FluoView analysis software. De Novo software version FCS Express 6 was used for flow cytometry analysis. One way analysis of variance (ANOVA), and student unpaired t-test were used to test statistical difference between two groups. Bonferroni's test was chosen as a post hoc test. Data were represented as mean ± standard deviation (mean ± SD) when biological independent replications was three to six (n =3-6) and significant difference value was less than 0.05 (P < 0.05).Figure , hiPSC cell lines were cultured in E8 medium for four days prior to induction of cardiac differentiation in order to generate a confluent cell layer Figure B.Monolayer cultures are easily accessible to most laboratories that are not equipped with suspension bioreactor technologies. Therefore, our priority aim was to standardize hiPSC-CMs differentiation under monolayer conditions to achieve high reproducibility, differentiation efficiency and CM yield. Following the protocol depicted in Figure ). CHIR99021 concentration range was chosen based on a titration experiment with increasing concentrations of CHIR99021 indicating that increasing CHIR99021 concentration to 10 and 12 μM did not further enhance differentiation. However, the efficiency of hiPSC-CM differentiation was reduced to 50.9 ± 17.0% and 61.9 ± 24.2% (n = 4) at CHIR99021 concentrations of 2 and 4 μM, respectively .The treatment of three different hiPSC lines  for the initial 24 h with CHIR99021 at the concentration of 6 to 8 μM followed by treatment with IWP2 and XAV939 from days 3 to 5 was sufficient to reach a maximal cardiomyocyte induction with >90% TNNT2-positive hiPSC-CMs on day 18 of differentiation ((Movies S1a and S1b) and were easily observed by naked eye without microscopic magnification from day 8 onwards Movie . This protocol is referred to as standard protocol in the following sections.Cardiomyocyte contractions were first observed by light microscopy at day 7 of differentiation Figure ). The total cardiomyocyte yield in these differentiations was 0.42 ± 0.07 x 106 per cm2 growth area (n=5) for NP0040, 0.41 ± 0.04 x 106 per cm2 growth area (n=4) for IMR90, and 0.34 ± 0.12 x 106 per cm2 growth area (n=3) for NP0141 iPSC lines Figure D.Flow cytometric analysis revealed that cardiac differentiation of NP0040, IMR90, and NP0141 iPSC lines in monolayer cultures yielded cell preparations containing 91.2 ± 0.7% (n = 5), 91.3 ± 1.6% (n = 4), and 88.6 ± 1.6% (n = 3) TNNT2-positive CMs, respectively  and addition of 8 μM CHIR99021 for the initial 24 h, but lack of treatment with IWP2 and XAV939 during days 3-5 of differentiation, poorly induced cardiomyocyte differentiation  , respectively . The most efficient and robust differentiation was achieved by combining IWP2 and XAV939 for 48 hours from day 3 to day 5 with continuous ascorbate supplementation throughout the whole differentiation process Figure A,B. All three factors synergistically increased the efficiency and reproducibility of cardiomyocyte differentiation to 90.2 ± 2.2% (n = 6) Figure A,B. The total cardiomyocyte yield was 0.51 ± 0.14 ×106 CMs per cm2 growth area when ascorbate, IWP2 and XAV939 were combined (n=6)  with various passage numbers of NP0040 hiPSCs, and different combinations of small molecules IWP2 and XAV939 and ascorbate were compared. 6 cells per cm2 of NP0040 cell line were used and induced to differentiate to hiPSC-CMs. At day 20 the fraction of TNNT2 positive cells was analyzed by flow cytometry in four biological independent replications. Representative data from these analyses are shown in Figure . Quantification of CM-purity indicates that 59.5 ± 9.6% (n = 4) and 56.4 ± 14.1% (n = 4) of TNNT2-positive cells were obtained at iPSC seeding densities of 0.3 and 0.4x106 cells per cm2, respectively. These values differed significantly (P < 0.05) from TNNT2 positive cells obtained in differentiation experiments starting with a seeding density of 0.01x106 cells per cm2  . In addition the maximally achievable CM yield per cm2 of culture substrate (“yield max”) was approximated by nonlinear regression: 0.1 ± 0.06x106 CMs/cm2 (n = 4) was yield max for seeding density of 0.02 x 106 hiPSCs / cm2 .Previous protocols show variations in the differentiation efficiency when suboptimal densities of PSCs are used in the initial stages of the differentiation protocol 6 cells per cm2, and again the CM purity and the yield were determined by flow cytometry in all nine groups. Flow cytometric analysis performed at day 18 of differentiation revealed that the purity and yield of CMs produced by our protocol are less dependent on the initial iPSC seeding density. The CM purity assessed as the fraction of TNNT2-positive cells ranged from 78.1 ± 14.3 % (n = 5) in the 0.01×106 cells/cm2 group to 87.8 ± 5.5 % (n = 5) in 0.08×106 cells/cm2 group  in the fraction of TNNT2-positive cells between all nine seeding densities (n=5)  were initially plated per cm2 . In order to determine the most efficient conditions, a sigmoidal curve was fitted based on the average cardiomyocyte yield to determine the yield max in this experiment (n = 5). This approximation revealed a maximal yield of 0.48 ± 0.17 (n = 5) ×106 CMs per cm2 growth area for all initial cell seeding densities in the range of 0.08 - 0.4×106 cells/cm2, and indicated that increasing the starting cell number beyond a certain value does not result in higher cardiomyocyte yields Figure C.In order to determine the effect of the hiPSC seeding density on cardiac differentiation efficiency in our optimized protocol, NP0040 hiPSCs were seeded at day -4 of differentiation at densities of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3 and 0.4 × 10Table S1). In this period, 5 μM of ROCK inhibitor was present in the medium for the first two days to increase the cell survival and support the formation of EBs Figure A. Employing the standard protocol as optimized for 2D monolayer differentiation cultures to the 3D bioreactor culture without further modification resulted in formation of EBs that exhibited spontaneously contracting areas already at day 7 of differentiation (Movie S3 and S4). To determine cardiomyocyte purity, flow cytometric analysis was performed, and analyses demonstrated that dissociated EBs were composed of 87.2 ± 0.9 % (n = 3) TNNT2-positive CMs at day 18 when an initial inoculum of 40 x 106 hiPSCs was used . In addition, cardiomyocyte-yield determined as the total number of TNNT2-positive cells in three independent differentiations, was 71.9 ± 29.8 ×106 CMs per 100 mL bioreactor culture volume for NP0040 hiPSC line Table .Cardiac bodies from suspension bioreactors were dissociated into single cells, and single cells were cultured on Matrigel-coated 48 wells plates to form monolayers of hiPSC-CMs. After two days of culturing the mono layer hiPSC-CMs were contracting macroscopically similar to hiPSC-CMs generated in the 2D system . Ventricular myosin light chain isoform MLC-2v was found to be highly expressed on day 10 , and less expressed on day 20  and onwards . Expression of the atrial isoform MLC-2a continuously increased until day 40 , and decreased on days 50  and 60  .We performed a 60 days follow up experiment with probe sampling every ten days to gain knowledge on the expression kinetics of subtype specific myosin light chain 2 isoforms. Cardiac proteins TNNT2, α-ACTININ, MLC-2v, and MLC-2a were detected by flow cytometry. Flow cytometric analysis indicated that pan-cardiomyocyte markers TNNT2 and α-ACTININ were expressed at low levels on day 10 , and highly expressed on day 20  which remained at the similar level until day 60  as described previously To further characterize the hiPSC-CMs we analysed the expression level of heart specific genes using genome wide DNA microarrays on day 20 of differentiation. It is expected that several heart specific genes such as genes essential for heart contraction would indicate high expression level. Table ls Table  and anal2+ transients was examined in the absence (Video S6) and presence of adrenergic stimulation by 10µM Iso (Video S7). In day 20 hiPSC-CMs, 10 µM Iso abolished spontaneous contractions, which coincided with the dispersion of spontaneous, global Ca2+ transients into high frequency local Ca2+ release events (Video S7), accompanied by an increase in cytosolic resting [Ca2+] . In contrast, application of Iso at day 30 and 40 did not interfere with the presence of global Ca2+ transients but led to an increase in beating frequency. A statistically significant difference (P < 0.001) was found for the increase in spontaneous beating frequency (beats per minute) between day 20 , 30  and 40 , as well as between untreated and Iso-treated samples at day 30  and day 40 . To check whether the kinetics of Ca2+ release and the responsiveness towards adrenergic stimulation increased with maturation, the calcium transients of hiPSC-CM recoded in the absence and presence of 10 µM Iso were analyzed for the time-to-peak (ttp) parameter . In both, day 30 and day 40 cells, Iso led to an acceleration of Ca2+ release kinetics: ttp = 436±60 ms (n=9) vs 332±54 ms (n=7) at day 30 and ttp = 403±17 ms (n=10) vs 280±31 ms (n=10) at day 40.Calcium ions play important roles in cellular physiology and its contribution in cardiomyocyte physiology can be utilized as a parameter to determine levels of cardiomyocyte maturation at different time points of the differentiation process. Therefore, we performed confocal calcium imaging of hiPSC-CMs on day 20, 30, and 40 of differentiation. The effect of prolonged culture of hiPSC-CMs on frequency and kinetics of contraction-related, global CaFigure 6, application of Iso induced significant increase of beating activity  compared with control beating rate  of hiPSC-CMs from 2D and 3D systems. The application of CCh evoked a significant reduction of beating frequency in both 2D and 3D hiPSC-CMs . These results revealed no differences between both 2D and 3D differentiation systems and suggest the presence of intact of ß-adrenergic and muscarinic signaling pathways .Furthermore, as one of the important factors of normal cardiac function is the intact response to different stimulations, we evaluated effects of Iso and muscarinic cholinergic receptor antagonist carbachol  on beating activity of hiPSC-CMs generated under 2D and 3D. This experiment was conducted to compare the quality of CMs generated by these different approaches. As shown in To determine the maturation status we recorded optical action potentials of monolayers of hiPSC-CMs at 25 and 33 days of differentiation. At 25 days the hiPSC-CMs showed spontaneous phase 4 depolarization but not at 33 days after differentiation Figure A. SpontaThe protocol provided in this study allows for robust and cost-efficient generation of hiPSC-CMs by continuous supplementation of ascorbate in the differentiation medium and combined treatment with IWP2 and XAV939 during day 3 to 5 of differentiation. We showed that this triple combination increases the efficiency and reproducibility of CM differentiation. Furthermore, our data demonstrates that combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling with IWP2 and XAV939 results in lower sensitivity of the cardiac differentiation efficiency to initial cell seeding density in monolayer differentiation cultures. Starting from hiPSC seeding densities in a broad range, differentiation was initiated after four days of subsequent culture without further controlling the confluency of the hiPSCs. This achievement is the most important innovation in the present protocol, because it substantially simplifies the differentiation process and eliminates the need to carefully control cell confluency. In contrast to common protocols, insulin was not supplemented throughout the whole differentiation process, thereby, in combination with use of homemade E8, reducing the expenses of the differentiation process.2 for coating hiPSC culture plastic ware, and (4) using a concentration of 8 µM CHIR99021 without titration.IWP2 attenuates porcupine Wnt signaling XAV939 is a potent tankyrase pathway inhibitor that causes cytoplasmic stabilization of the axin protein Figure 8): On one hand, activation of the Wnt canonical pathway leads to release of β-catenin from axin - glycogen synthase kinase 3 β protein complex, allowing β-catenin to translocate into the nucleus and facilitate maintenance of the stem cells pluripotency network Wnt signaling has a biphasic property on stemness and CM differentiation (hypothetic interplay of signaling pathways: Combining either IWP2 or XAV939 with continuous ascorbate supplementation increased cardiac fate decision in comparison to groups where no ascorbate was added. Ascorbate seems to enhance the CM differentiation process independently of its antioxidative properties Cardiomyocytes can be differentiated from human PSCs, either in monolayer or in suspension cultures Secondly, scalable suspension bioreactor differentiation is not easy reproducible in each laboratory due to technical challenges such as optimizing agitation speed Despite of overcoming limitations by fluctuations in initial cell seeding density, cell line variations, difficulties in scaling up from 2D to 3D culture systems, reducing high cell culture expenses, and batch to batch variations in hiPSC-CM differentiation, still further investigations are required to find optimal cell dissociation routines and replace Matrigel. Removing Matrigel for coating is mandatory in translational approaches and it is advisable to utilize recombinant laminins instead.Our data is in line with previously published protocols showing that hiPSC-CMs express several human CM-specific markers including TNNT2, α-actinin, MLC-2v, and MLC-2a, as well as proving physiological calcium transients, and responses to beta adrenergic receptor stimulation In summary, temporal manipulation of Wnt signaling via combined inhibition of porcupine and tankyrase pathways with continuously ascorbate supplementation is an effective method for cardiomyocyte differentiation, in both monolayer and suspension culture conditions with less cost, low variabilities, and >90%TNNT2 positive cells. This method should ease the translation of basic research to large animal experiments to pave the road for clinic trials and allow more laboratories to adapt the differentiation technology in reasonable time.Supplementary Figures, Table S1.Click here for additional data file.Supplementary Tables S2-S3.Click here for additional data file.Supplementary Movies.Click here for additional data file.


Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.Nontypeable  Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi-specific candidate vaccine antigen.Nontypeable  Haemophilus influenzae (NTHi) are composed of multiple heterogeneous glycoforms (–N-acetyl-5-neuraminic acid (Neu5Ac) (in vivo and in vitro (–15––N-acetyllactosamine (The lipooligosaccharides (LOS) of nontypeable ycoforms . This isycoforms . In addiycoforms . In the ycoforms –8. LOS g(Neu5Ac) , 10. Man(Neu5Ac)  and is ain vitro . NTHi cain vitro –15. The itro –15– and is pitro –15––21. Bothtro –15––. SiaA sitosamine , and likN-acetylhexosamines and Neu5Ac , suggesting that this enzyme may be able to substitute KDO for Neu5Ac  of MAb 6E4 to KDO was 11.5 ± 1.1 nM, which was 300-fold better binding than to Neu5Ac or Neu5Gc. No binding was detected with the trisaccharides tested.To confirm the specificity of monoclonal antibody (MAb) 6E4, surface plasmon resonance (SPR) was used to test the binding specificity of MAb 6E4 binding to KDO monosaccharide. MAb 6E4 was immobilized on a series S sensor chip CM5. Five sugars were tested, KDO, Neu5Ac, Neu5Gc, and the trisaccharides, α2-3 sialylactosamine (2-3SLN) containing either an acetyl or glycolyl group. As can be seen in Studies in our laboratory have previously shown that MAb 6E4 derived from immunizing mice with NTHi 2019 binds to a terminal LOS epitope containing KDO  Fig. 1)Fig. 1). N-acetyllactosamine in the presence of 100 µM Neu5Ac  containing either an acetyl group (2-3SLN-AC) or glycolyl group (2-3SLN-Gc). Download Copyright © 2018 Apicella et al.2018Apicella et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01401-18.2FIG S2 ΔsiaA1 mutant (lanes 4 to 6), ΔsiaA1 Δlic3B mutant (lanes 7 to 9), ΔsiaA1 Δlic3A ΔlsgB mutant (lanes 10 to 12), and ΔsiaA1 Δlic3A ΔlsgB::lic3B mutant (lanes 13 to 15). Extracts in lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, and 14 were from organisms grown in the presence of 100 μg Neu5Ac. Extracts in lane 2, 5, 8, 11, and 14 were from organisms treated with sialidase prior to electrophoresis. Extracts in lanes 3, 6, 9, 12, and 15 were from organisms grown in the absence of Neu5Ac. Lanes 16 contain LOS from N. gonorrhoeae PID 2 as a control in which a 4.5- and a 5.0-kDa band bind MAb 3F11. These blots demonstrate that MAb 6E4 did not react with any extracts derived from strains grown in the presence of Neu5Ac and that only extracts containing a functional LsgB reacted with Mab 6E4. In addition, the bottom blot shows that MAb 3F11 reactivity is reduced when 6E4 binding occurs. Finally, binding of both 6E4 and 3F11 MAbs does not occur to the LOS in extracts of organisms grown in the presence of Neu5Ac in the absence of sialidase treatment. Download FIG S2, TIF file, 3.1 MB.Results of two Western blots of studies with proteinase K extracts of NTHi strain 2019 (lanes 1 to 3), Copyright © 2018 Apicella et al.2018Apicella et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lsgB. lsgB gene reacted with MAb 6E4. In addition, MAb 3F11 reactivity is reduced when MAb 6E4 binding occurs. These data indicate that the LsgB sialyltransferase has the capacity to utilize CMP-KDO as a substrate in the absence of CMP-Neu5Ac. In addition, LsgB can place KDO onto the terminal LOS acetyllactosamine of NTHi strains when the organisms are grown in the absence of Neu5Ac.We constructed a series of deletion mutant combinations in all four sialyltransferases and in all combinations of the sialyltransferases in strain NTHi 2019  on solid and liquid media and in the presence and absence of Neu5Ac. NTHi strain 2019 was studied after 12 h of growth on sRPMI agarose plates or in sRPMI broth. RNA was isolated, and the results are shown in lsgB is increased approximately sevenfold when the organisms are grown on solid medium compared to organisms grown in broth  the bactericidal effect of MAb 6E4. Download FIG S3, TIF file, 3.7 MB.MAb 6E4 bactericidal effect is directly related to the presence of Neu5Ac in the NTHi strain 2019. Neu5Ac was sequentially increased in the growth medium from 0.1 to 100 µg/ml. Growth of NTHi in the presence of greater than 1 µg of Neu5Ac significantly inhibits  (NTHi cannot synthesize Neu5Ac and must acquire it from its environment . Previougulated) , 17, 27.gulated) . NTHi dogulated) .lsgB activity is suppressed when the organism is grown in liquid media with an ~7-fold difference in expression comparing growth in solid media growth to growth in broth. siaR appears to play a role in this difference, as it disappears when siaR is mutated. The biological significance of this is not clear, but the in vivo studies showing expression of the KDO epitope in infected tissue may suggest that plate growth better simulates the in vivo environment than in vitro growth in broth.It is interesting that N-acetyllactosamine conjugated to an immunogenic protein may be an approach to develop a component of a multicomponent vaccine similar to Bexsero or Trumenba used for protection against the serogroup B meningococcus. Additionally, the KDO epitope offers the ability to potentially use a sugar-based vaccine for NTHi without concern about cross-reactivity with human tissues, since KDO is found only in Gram-negative bacteria. Also, its effect on commensal Gram-negative bacteria would not be a concern, since KDO is buried in the deep LPS core and not available to be bound by MAb 6E4  of the strains showed significant killing after exposure to MAb 6E4. Given the difficulties in developing a successful vaccine to NTHi, using a glycoconjugate composed of either multiple KDOs or a KDO- MAb 6E4 , 23.N-acetyllactosamine glycoform in vivo and the possibility that this may be a target for bactericidal antibody opens the possibility of developing a multicomponent vaccine to NTHi.These studies further demonstrate the unique biology of NTHi LOS. The observation that the LsgB sialyltransferase is responsible for placing a terminal KDO residue on a 2. RPMI 1640  was used as a sialic acid-free chemically defined medium. Supplemented RPMI 1640 (sRPMI) was prepared with protoporphyrin IX (0.5 µg/ml), β-NAD (10 µg/ml), hypoxanthine (0.1 mg/ml), uracil (0.1 mg/ml), and sodium pyruvate (0.8 mM). Neu5Ac was added as indicated. In addition, 34 NTHi strains from different clinical conditions were studied in the whole-cell ELISA assay. These 33 strains have been previously described . PCR was performed using primers obtained from Integrated DNA Technologies . Primers were labeled with 6-carboxyfluorescein. A modified version of the capillary electrophoresis fragment length analysis developed by Fox et al. . Colonies in which the gene of interest was predominantly 90% phase on based on capillary electrophoresis studies were selected for further study.Deletions of lsewhere . To prodN-acetyllactosamine epitope  6E4 is a murine IgG3 monoclonal antibody that has been previously described , 23, 25. epitope . MAb 2C3eisseria , 34. Foreisseria .N-glycolylneuraminic acid (Neu5Gc) was reduced to 50 µM. Analysis was carried out using the Biacore T200 evaluation software.The 6E4 antibody was immobilized onto a series S CM5 sensor chip via amine coupling using the amine coupling wizard template on a Biacore T200 system. Flow cell 1 was made as a blank cell (ethanolamine-blocked surface) to allow for double reference subtraction. Sugars were initially flowed over the 6E4 antibody from 0.01 to 100 µM, and the concentration for each sample was adjusted on the basis of observed binding as previously described . KDO wasp-nitrophenylphosphate was applied at a concentration of 1.0 mg/ml. Absorbance was read in a Tecan Model Infinite 200 Pro plate reader at 405 nm.A whole-cell enzyme-linked immunosorbent assay was performed using monoclonal antibodies 6E4 and 3F11 by the method of Abdillahi and Poolman . Bacteriin vivo, we embedded the biofilm sample in an OCT compound  as previously described  in neuraminidase buffer  at 37°C for 2 h. Western blotting was performed by the method of Blake et al.  sodium dodecyl sulfate, pH 6.8. Proteinase K was added to a final concentration of 50 µg/ml, and samples were incubated overnight at 37°C. After three ethanol precipitations, samples were treated with DNase I and RNase A. Samples were then phenol extracted, ethanol precipitated three more times, and centrifuged at 120,000 × e et al. .Cell samples were homogenized by pipetting with 1 ml of TRIZOL total RNA isolation reagent . Lysates were transferred to Lysing Matrix B tubes  and vortexed for 60 s. The lysates were extracted with 200 µl of chloroform and centrifuged for 15 min. The supernatant was precipitated with 1 volume of isopropanol for 10 min at 25°C (room temperature). RNA was pelleted and washed with 75% ethanol, and the pellet was dried at room temperature. RNA was reconstituted in ultrapure water and cleaned with a Qiagen RNeasy minikit . The RNA was then treated with reagents in the TURBO DNA-free kit  for 30 min at 37°C and purified using a phenol-chloroform-isoamyl alcohol  extraction, followed by an ethanol precipitation. One microgram of RNA was reverse transcribed using SuperScript II reverse transcriptase (Life Technologies). SYBR green quantitative real-time PCR (qRT-PCR) was used to measure gene expression. For each target, qRT-PCR primer sets were selected using Primer Express software  and obtained from Integrated DNA Technologies . Relative RNA quantities were determined by standard curve (fivefold dilutions of purified genomic DNA [gDNA] ranging from 100 to 0.032 ng/µl), and all values were normalized to the amount of outer membrane protein 6 (OmpP6) RNA in each sample. OmpP6 is considered constitutively expressed in NTHi. Our data on more than 60 NTHi mRNA samples confirmed this fact. We performed 20-µl reactions in triplicate in 1× Kapa SYBR Fast qPCR master mix  with 4 ng of template or gDNA standard and a final concentration of 250 nM for each primer. qRT-PCR was performed on an ABI Prism 7900 sequence detection system (Life Technologies). The data were normalized against NTHi P6 and standardized against genomic DNA from wild-type NTHi 2019 strain.t tests were used to compare differences in ELISA, bactericidal, and RT-PCR results. Values that met a P value cutoff of <0.05 were considered statistically different.Statistical analysis was performed with Prism 7 software . Student’s


To provide consistent 3DP processing from the formulated feedstocks, the carefully chosen dispersant performed the double function of adjusting the overall viscosity of the photopolymer and provided good matrix-to-filler bonding. Depending on the ceramic powder content, the optimal viscosities for reproducible 3DP with resolution better than 100 µm were η(TO) = 1.20 ± 0.02 Pa.s and η(CCT) = 0.72 ± 0.05 Pa.s for 20% w/v TO/resin and 20% w/v CCT/resin composites at 0.1 s−1 respectively, thus showing a significant dependence of the “printability” on the dispersed particle sizes. The complex dielectric properties of the as-3D printed samples from pure commercial photopolymer and the bespoke ceramic/photopolymer mixes are investigated at 2.5 GHz, 5 GHz, and in the 12–18 GHz frequency range. The results show that the addition of 20% w/v of TO and CCT ceramic powder to the initial photopolymer increased the real part of the permittivity of the 3DP composites from ε’ = 2.7 ± 0.02 to ε’(TO) = 3.88 ± 0.02 and ε’(CCT) = 3.5 ± 0.02 respectively. The present work can be used as a guideline for high-resolution 3DP of structures possessing high-ε.The formulation of a high dielectric permittivity ceramic/polymer composite feedstock for daylight vat photopolymerization 3D printing (3DP) is demonstrated, targeting 3DP of devices for microwave and THz applications. The precursor is composed of a commercial visible light photo-reactive polymer (VIS-curable photopolymer) and dispersed titanium dioxide (TiO Additive Manufacturing (AM), also known as three-dimensional printing 3DP), has shown the potential to fabricate components with tailored and complex designs and is being adopted in various industrial domains. The American Society for Testing and Materials (ASTM) classified AM into 7 standard processes . Among tDP, has sVarious 3DP processes have been extensively used in the fabrication of optical , THz 6,,6,7, micThe present work was mainly motivated by the need for a material exhibiting high dielectric properties suitable for 3DP of devices for high-frequency applications, targeting the GHz–THz range. While, as shown in the example above, material extrusion can provide feedstock with dielectric permittivity, ε, as high as 11, and feature resolution is typically within the 100–200 µm range. In the present study, the Digital Light Processing  was chosen due to its ability to provide smooth surface finishes  and highw/w CNT-loaded composite increased almost 3 orders of magnitude compared to the pure photopolymer formulation.Recently, the combination of structural (polymers) and functional (conducting and high permittivity ceramic fillers) materials has been investigated to achieve multifunctional 3DP parts with good structural stability . Furtherw/w of SiO2 nano-powder into a commercial SLA feedstock increased the tensile strength and modulus of the 3DP samples by 20.6% and 65.1% respectively. Lin et al. [w/w graphene oxide leading to a 62.2% increase of the tensile strength. Manapat et al. [w/w graphene oxide; similarly, adding 10% w/w multi-walled CNT into the SLA precursor resulted in 7.5% improvement in the tensile strength [w/w CNT composite in the 4—18 GHz frequency range [Weng et al.  have obsn et al.  3D printt et al.  reportedstrength . The diecy range  revealedAlthough there has been prior research in 3DP of structurally enhanced photopolymer/ceramic composites, there has been little investigation of Vat Polymerisation polymer/composites potentially suitable for novel electromagnetic applications, where the combination of high dielectric permittivity, low-dielectric loss, and good spatial resolution is essential. It should be noted that the application of 3DP technologies for fabrication of dielectric devices for frequencies above 20 GHz remain almost an unexplored area. Otter et al.  have dem2 (TO) and CaCu3Ti4O12 (CCT) are used as a high-ε ceramic fillers with the objective of achieving significant improvement in the dynamic mechanical, thermal, and dielectric properties of the DPP 3DP composites, as compared to off-the-shelf commercial feedstock, and to potentially increase the range of applications of this process.Here, we focus on the development of a feedstock precursor with a high dielectric constant for high-resolution low-cost Daylight Polymer Printing (DPP) technology. Nano- and micro-powders of TiO2), nanopowder, <100 nm  and calcium copper titanate (CaCu3Ti4O12), ~3–5 µm . Photopolymer containing 5, 10, 20% w/v filler (TO and CCT) along with 2% w/w dispersing agent (with respect to filler weight) composites were formulated.To formulate the photopolymer composite dispersions and to enhance the distribution of the ceramic powder in the photopolymer composite dispersion, a dispersing agent, DISPERBYK-2055  was added to the photopolymer (daylight precision firm resin (black) ) in a 100 mL amber glass bottle and stirred (1000 rpm) on a magnetic stirrer at RT for 1 h. After that, the filler (nano/micro-sized) was added to the previous solution and magnetically stirred (1000 rpm) at RT for 6 h and ultra-sonicated for 2 h. Fillers employed were titanium (IV) oxide  3D Printer  was used for the DPP 3DP of the formulated photopolymer. A diagram of the LCP 3D printer is shown in −1.Dynamic viscosity of the formulated photopolymers was measured using a HAAKE Mars III Rheometer  and 25 mm parallel plates at RT with a shear rate range of 0.01–1000 sX-ray diffraction (XRD) spectra of the two fillers were recorded using an Empyrean X-ray Diffractometer  with a CoKα source at a generator voltage of 40 kV, 40 mA current, wavelength of 1.79 Å, scan range of 10 to 110° and step size 0.01° at RT.Scanning electron microscopy (SEM) imaging was performed using a Zeiss Sigma FE SEM  on powder samples which were sputter coated using an Au/Pd target.Surface topography of the 3DP composites was obtained using a 3D Optical Profiler , white light interferometer with 50× optical lens. SEM imaging was carried out using a JEOL-SEM and Zeiss Sigma FE SEM  on fractured samples, which were sputter coated using an Au/Pd target.Thermal degradation behaviour of the 3DP composites was investigated using a TGA1-STARe Thermogravimetric analyser (TGA)  in the temperature range 25–550 °C, at a heating rate of 10 °C/min under nitrogen atmosphere. Differential Scanning Calorimetry measurements were carried out using a DSC 1 Stare  between −40 °C and 200 °C with a heating and cooling rate of 10 °C/min.Dynamic Mechanical Thermal Analysis (DMTA) was performed on samples 5 mm × 9 mm × 1 mm in single cantilever mode with a 50 µm amplitude, a frequency of 1 Hz and a heating rate of 5 °C/min in the temperature range of −30–200 °C using a Tritec 2000 DMA . The frequency dependence of storage modulus (G′), loss modulus (G″) and loss factor (Tan δ) were measured for the same dimension samples in single cantilever mode with a 50 µm amplitude at 25 °C in the frequency range of 0.01–50 Hz.11, S22, and transmission S21 and S12 parameters, followed by retrieving the relative complex permittivity εr using the Nicholson–Ross–Weir method [The complex dielectric properties of the 3DP composites were obtained using a 2-port Vector Network Analyzer  at 2.5 GHz and 5 GHz with the split-post dielectric resonator (SPDR) technique and in the 12–18 GHz frequency range using the waveguide technique. When using the waveguide technique, the SPDR was used for accurate measurements of the dielectric loss . The SPDr method ,36.TO nanoparticles are spherical and agglomerated, while CCT particles show polyhedral morphology with a particle size of 3–5 µm a,b. The w/v TO increased from 0.30 Pa.s (unfilled photo-resin at 0.1 s−1) to 0.50 ± 0.02, 0.56 ± 0.02 and 1.20 ± 0.02 Pa.s at 0.1 s−1, respectively. It can be observed from w/v CCT composites increased to 0.37 ± 0.05, 0.49 ± 0.05 and 0.72 ± 0.05 Pa.s at 0.1 s–1, respectively. The increase in viscosity with increasing filler content is higher for the TO nano-composite than for the CCT micro-composite  is greater than the viscosity of composites filled with larger particles (micrometre range) at the same volume fraction ,45.Faitel’son et al.  preparedw/v TO (0.18 ± 0.02 µm) and CCT (0.32 ± 0.02 µm) 3DP composites is higher than that of the 3DP un-filled photopolymer (0.11 ± 0.02 µm), due to the presence of dispersed filler particles.w/v.w/v of TO and CCT, respectively. In general, the addition of TO appeared to be a more effective route for increasing the relative dielectric permittivity of the composites.w/v TO composites, the permittivity is ε′(TO) = 3.85 − j0.0321. The results of dielectric permittivity in TO-based 3DP samples are in good agreement with the effective medium theory [w/v volume fractions of TO and CCT. The predicted values were calculated in accordance with [n = 0.1 correction factor to compensate for the shape and diameter of the fillers) and CCT are less spherical (n = 0.12):effε, mε, and fε are the effective real part of the dielectric constant of the composite, polymer matrix, and ceramic filler, respectively, and f and n are the volume fraction and the correction. The permittivities fε of the TO and CCT ceramic filler were stood 100 and 65 respectively. Surprisingly, CCT-filled samples show very moderate increase in permittivity in spite of the reported giant dielectric constant in this material [The results of dielectric permittivity measurements obtained by the SPDR method for nominal 5 GHz are consistent with the waveguide measurements and show the complex dielectric permittivity of the 3DP un-filled photopolymer as ε′(resin) = 2.89 − j0.0197, while for the 3DP 20% m theory  and expem theory . Figure nce with   was performed for the 3DP TO and CCT composites to observe the effect of different fillers on their glass transition temperature, Tr chains .0) and maximum degradation temperature (Tmax) were decreased for the composites compared to the un-filled photopolymer. Furthermore, T0 gradually decreased with an increase in the composite’s filler content. This may be due to the decrease in molecular weight during photo-polymerization (DPP 3DP process) in the presence of fillers [The thermal degradation of the 3DP photopolymer and its composites presented in  fillers . The pri fillers . Table 1w/v CCT composites also significantly increased at 25 °C  compared to the pure photopolymer  = 3.88 ± 0.02 and ε′(CCT) = 3.5 ± 0.02 for the 20% w/v TO and 20% w/v CCT composites, compared to ε′(photopolymer) = 2.67 ± 0.02, and showed no dispersion in the 2.5–18 GHz frequency range. Such dielectric properties may be attractive for 3DP of advanced electro-magnetic devices for high-frequency communications, where high spatial resolution of the feature elements is essential. This paper can also be used as a guideline for the formulation of bespoke precursors with tailored properties  for DPP 3DP, with the optimal viscosities for reproducible 3DP in the range from 1.20 to 0.72 Pa.s for nano- and micro-sized functional inclusions, respectively.The present research explores the high resolution DPP 3DP of photopolymer composites containing TO and CCT particles based on visible light curing technology for high frequency electromagnetic applications. Composites filled with 5, 10 and 20%


Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. The present study characterized a previously unrecognized S. aureus toxin. This secreted toxin was purified from strain Newman ΔhlaΔhlg, the N-terminal sequence determined, the gene cloned, and the purified recombinant protein was tested in the rabbit cornea. The virulence of a toxin deletion mutant was compared to its parent and the mutant after gene restoration (rescue strain). The toxin (23 kDa) had an N-terminal sequence matching the Newman superantigen-like protein SSL1. An SSL1 homodimer (46 kDa) had proteolytic activity as demonstrated by zymography and cleavage of a synthetic substrate, collagens, and cytokines ; the protease was susceptible to serine protease inhibitors. As compared to the parent and rescue strains, the ssl1 mutant had significantly reduced virulence, but not reduced bacterial growth, in vivo. The ocular isolates tested had the ssl1 gene, with allele type 2 being the predominant type. SSL1 is a protease with corneal virulence and activity on host defense and structural proteins. Staphylococcus aureus causes infections in a wide variety of non-ocular sites and is a major ocular pathogen  with the expression vector pQE-60 was grown in LB medium with ampicillin 100 µg/mL and kanamycin 25 µg/mL. vitamins ,16. Anti2, and 1 µM ZnCl2 at 37 °C for 24 h. After staining with Coomassie blue, the area where proteolysis of gelatin occurred reveals clear (white) bands against a blue background.Gelatin (0.1%) zymograms were performed using 10% acrylamide gels under non-denaturing conditions (no SDS) or denaturing conditions (with SDS) and stained with Coomassie blue . BrieflyCulture supernatants were prepared, concentrated, and then fractionated by molecular sieve chromatography using Sephacryl S-100 . FractioΔhlaΔhlg, ocular isolate 295.236, or strain MW2 was used as template in PCR reactions. The forward primer was 5′-AACCATGGGTAAATTTAAAGCGATAGCA-3′ and the reverse primer was 5′-TTAGATCTTTTCATTTCTACTAGAAT-3′. The recombinant protein was secreted by E. coli. All DNA constructs were verified by sequencing . The recombinant protein was purified from culture supernatants by metal affinity  and Sephacryl S-100 chromatography.The gene was translated and the signal peptide cleavage site was identified using ExPASy’s software. For cloning into plasmid pQE-60, the NcoI and BglII restriction sites were incorporated into the PCR product. Genomic DNA of strain Newman n = 3) by methods described previously [Antibody to the recombinant Newman SSL1 protein monomer was produced in rabbits  was incubated for 2 h at 37 °C with 1 µg of recombinant IL-17A, IFN-γ, or IL-8  and analyzed by SDS-PAGE. Recombinant SSL1 was also incubated with fluorescent collagen type I or type IV  at 37 °C and the fluorescence (excitation at 485 nm and emission at 520 nm) was measured every 30 min for 6.5 h.405 in a microtiter plate reader every 10 minutes for 2 h. The Km and Vmax were determined by plotting the substrate concentration versus velocity using the GraphPad Prism 6 software. The optimum pH and temperature for activity were determined as described previously [2HPO4 buffer (pH 3–7), Tris buffer (pH 8–9), sodium bicarbonate buffer (pH 10–11), or potassium chloride - sodium hydroxide buffer (pH 12–13). Briefly, the substrate was added to a reaction mixture of SSL1 (10 µg) and a buffer of a certain pH and OD405 was measured every 30 min for a 2.5-h period at room temperature. Controls consisted of the substrate at the pH tested without SSL1. To determine the optimal temperature for SSL1 activity, purified recombinant SSL1 (5 µg) was incubated with the substrate CPL at various temperatures ranging from 5 °C to 65 °C for 5 h. After incubation, OD405 was measured. Controls consisted of the substrate at the temperature tested without SSL1.Chromozym PL  was used to determine SSL1 susceptibility to protease inhibitors. The substrate CPL mixed with enzyme samples was assayed in triplicate in a 96-well plate at 37 °C. Optical density (OD) at 405 nm was measured. Samples without enzyme or with heat-inactivated enzyme served as negative controls. The enzyme kinetics determination was performed by incubating SSL1 (20 µg) with varying concentrations of the substrate CPL at 37 °C and measuring ODeviously . The optssl1 gene of the S. aureus isolates was amplified by PCR and sequenced in both directions . A pair of universal flanking primers was used: 5′-ATAGATTGGGGCTAAAAATT-3′ and 5′-ATAGCGCTTTGTTGTTAGAAAGTA-3′. PCR reactions were performed using 0.1 µg S. aureus genomic DNA, 50 picomoles of each primer, and the GoTaq green master mix from Promega . The cycling conditions were as follows: 94 °C for 1 min, then 30 cycles of 94 °C for 20 s, 54 °C for 20 s, and 68 °C for 45 s. The resulting ssl1 gene sequences were compared to the allele type sequences as described by McCarthy and Lindsay [The  Lindsay . ΔhlaΔhlg double mutant (EryR/TetR) was created by phage transduction of the antibiotic markers from S. aureus strain 8325-4 ΔhlaΔhlg with phage 85. To create the triple mutant (Newman ΔhlaΔhlgΔssl1), the ssl1 gene was deleted from the chromosome of the double mutant by allelic exchange without an antibiotic marker. A construct for the unmarked deletion of ssl1 was made by SOE-PCR [The Newman  SOE-PCR . An upstTCTAGAATATTCATCAATTCTGGTAAATATGG-3′ XbaI; B primer 5′-CATAATTTTTAGCCCCAATCTATTTAAATTTG-3′) and downstream CD fragment  were amplified and joined by PCR using Phusion polymerase . The product was digested with XbaI/PstI and ligated into pIMC5 digested with SpeI/PstI and transformed into E. coli DH10B. The 2-kb insert was verified by sequencing. The construct pIMC5-ssl1 was transformed into S. aureus RN4220 and then transduced into Newman ΔhlaΔhlg with phage 85. Allelic exchange was conducted as described previously [ΔhlaΔhlgΔssl1 mutants screened with OUT primers (5′-TAATTAATACGACCATCGCCAGC-3′ / 5′-TCGTCATCAAGTATATGTGTTATGC-3′). To restore the ssl1 gene in the Newman ΔhlaΔhlgΔssl1 background, the ssl1 gene and flanking sequence were amplified from Newman genomic DNA with the above A and D primers. The product was cloned into pIMC5 producing pIMC5ssl1+ and allelic exchange performed in the Newman ΔhlaΔhlgΔssl1 background yielding Newman ΔhlaΔhlgΔssl1/ssl1.(A primer 5′-TATAeviously  with potS. aureus in 10 µL tryptic soy broth (n ≥ 10 eyes/group) [The use of animals, New Zealand white rabbits (2.0–3.0 kg) of either sex, adhered to the protocol (1060D) approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Rabbits were anesthetized by subcutaneous injection of a mixture of xylazine  and ketamine . Proparacaine  was topically applied to the eyes prior to intrastromal corneal injections (10 µL) of recombinant toxin, concentrated supernatant (50-fold), or 100 CFU of s/group) . Rabbit eyes underwent slit lamp examination scoring (SLE) as described by Arana et al. . Brieflyt tests between least-squared means from each group were performed. P ≤ 0.05 was considered significant. Mean and standard error of the mean (SEM) were calculated using Microsoft Excel. Statistical analyses of SLE scores and CFU determinations were performed using SAS software . For SLE


Aspergillus ochraceus was isolated from the Mediterranean sponge Agelas oroides. The initial fermentation of the fungus on solid rice medium yielded 16 known compounds (4–19). The addition of several inorganic salts to the rice medium mainly influenced the accumulation of these secondary metabolites. Fermentation of the fungus on white bean medium yielded the new waspergillamide B (1) featuring an unusual p-nitrobenzoic acid as partial structure. Moreover, two new compounds, ochraspergillic acids A and B (2 and 3), which are both adducts of dihydropenicillic acid and o- or p-aminobenzoic acid, were isolated from the co-culture of the fungus with Bacillus subtilis. Compound 2 was also detected in axenic fungal cultures following the addition of either anthranilic acid or tryptophan to the rice medium. The structures of the new compounds were established by 1D and 2DNMR experiments as well as from the HRMS data. The absolute configuration of 1 was elucidated following hydrolysis and derivatization of the amino acids using Marfey’s reagent. Viomellein (9) and ochratoxin B (18) exhibited strong cytotoxicity against the A2780 human ovarian carcinoma cells with IC50 values of 5.0 and 3.0 µM, respectively.The fungus  Acremonium chrysogenum as well as the potential anticancer drug plinabulin which is derived from the fungal metabolitehalimide obtained from a marine Aspergillus sp. D23 +22 ; UV (MeOH) λmax 267 nm; 1H and 13C NMR data, m/z 451.1824 [M + H]+ .Waspergillamide B (2): yellow oil; UV (MeOH) λmax 353, 254, and 221 nm; 1H and 13C NMR data, m/z 308.1134 [M + H]+ .Ochraspergillic acid A (3): yellow oil; UV (MeOH) λmax 306 and 224 nm; 1H and 13C NMR data, m/z 308.1127 [M + H]+ .Ochraspergillic acid B  were added to the first aliquot of 1, while 100 µL of a 1% acetone solution of D-FDAA were added to the second aliquot of 1. The mixtures were kept at 40°C for 1 h and occasionally shaken by hand. To stop the reaction, 20 µL of 1M HCl were added. The mixtures were centrifuged at 13,000 rpm for 10 min and an aliquot of 10 µL was analyzed using an RP-HPLC column with UV detection at 340 nm. The retention times of the L- and D-FDAA product of 1 were compared to each other. A Macherey-Nagel EC250/4.6 NUCLEOSIL250-5 C4-column  was used for HPLC analysis. The gradient of MeOH-H2O was changed from 15% to 60% MeOH over 55 min at a flow rate of 1 mL/min  and cisplatin for A2780 (IC50 2.2 µM) were used. As negative controls,0.1% ethylene glycol monomethyl ether in DMSO was used for L5178Y, whereas0.9% NaCl, 0.1% DMSO, and 1% DMSO were used for A2780.Cytoxocicity was investigated using the MTT -2,5-diphenyltetrazolium bromide) method against the mouse lymphoma cell line L5178Y or the human ovarian carcinoma cell line A2780 as previously described ,42. As p