Cupriavidus necator is a rare example of a truly O2-tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e− and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6[4Fe–3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5[4Fe–4S] or Cys4[4Fe–4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions.The membrane-bound [NiFe]-hydrogenase of  2-tolerant [NiFe] hydrogenase was transformed into different [4Fe-4S] clusters, and structural, spectroscopic and electrochemical analyses of the enzyme variants revealed an O2-tolerance mechanism at various levels.The [4Fe-3S] cluster of an O Depending on the metal center composition, these metalloenzymes are classified as either [FeFe]- or [NiFe]-hydrogenases.2,3 Consistent with their anaerobic origin, hydrogenases are commonly highly susceptible to molecular oxygen, which either inactivates or even destroys the enzymes. Thus, the emergence of the oxygenic atmosphere posed evolutionary pressure towards a microbial metabolism that also functions under oxic conditions. Although the availability of free H2 declined dramatically during these times, some bacterial species evolved O2-tolerant [NiFe]-hydrogenases that feed the H2-derived electrons into the quinone pool of the aerobic respiratory chain.4 A prominent member of these so-called Knallgas bacteria is Cupriavidus necator H16 , which employs four different O2-tolerant [NiFe]-hydrogenases for generating cellular energy from the controlled oxidation of H2 in the presence of O2.5 Among them, only the tripartite membrane-bound [NiFe]-hydrogenase (MBH) is directly linked to the respiratory chain.6 The MBH large subunit contains the catalytic center composed of a nickel and an iron ion, which are coordinated to the protein by four Cys-derived thiolates. One CO and two cyanide ligands additionally ligate the iron  experiments.9 According to a current model, the proximal [4Fe–3S] cluster contributes to efficient O2 detoxification in the catalytic center, i.e., rapid and complete reduction of O2 to 2H2O with 4e− and 4H+ previously obtained from H2 oxidation .3,22,25This unusual trait is supported by a plethora of biochemical, electrochemical, spectroscopic, computational, and crystallographic results obtained for MBH enzymes from xidation .9,15 In ablished . Notably2 oxidation activity under aerobic conditions, and the proximal [4Fe–3S] cluster appears to play a crucial role in this protective mechanism. In this context, C. necator MBH variants bearing cysteine exchanges at the proximal [4Fe–3S] cluster were analyzed under various oxidizing and reducing conditions by X-ray crystallography, biochemistry, protein film electrochemistry, spectroscopy, and spectro-electrochemistry to investigate the role of the two supernumerary cysteines at the proximal cluster. The results are of great importance for a generalization on the origin of O2-tolerance of [NiFe]-hydrogenases.Prevention of any oxidative damage of the active site is a prerequisite for sustained HC. necator MBH derivatives carrying exchanges of cysteine for glycine at the positions 19 and/or 120 in the small subunit HoxK, which are hereafter referred to as MBHC19G, MBHC120G and MBHC19G/C120G. While the native MBH and the MBHC19G protein variants showed similar yields, approx. half the amount was obtained for MBHC19G/C120G  spectro-electrochemistry as well as infrared (IR), resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy, to correlate activities with the structural/electronic properties of the metal centers.To obtain a detailed view of the proximal cluster architecture, all three engineered MBH variants were analyzed by protein X-ray crystallography. Additionally, the two MBHC19G, MBHC120G and MBHC19G/C120G variants were crystallized under either aerobic/oxidizing or anaerobic/H2-reducing conditions. The crystal structures were solved by molecular replacement, and the models were refined to resolutions ranging from 1.60 Å to 1.93 Å (PDB entries 8POU-8POZ). To qualify positive peaks in the mFo-DFC electron density maps, anomalous diffraction data were collected.The MBH2-reduced MBHC19G diffracted to resolutions up to 1.61 Å and 1.60 Å, respectively  position, previously observed for the Fe3 in the native [4Fe–3S] cluster. Approximately 80% of Fe3 were shifted by 1.4 Å to a position corresponding to a cubane corner (Fe3′), thereby forming a bond to S4 instead of Cys120   were located at 2.5 Å distance from the thiolate group of Cys19, which was also at a bond distance to Fe1 and Fe4  revealed the proximal Fe–S cluster in a similar structure/composition as in the reduced protein, except that Fe4 showed conformational flexibility  with Glu76 of the MBH small subunit. This particular Fe4′ position differs by 0.6 Å from the classical Fe4 position of native MBH in the super-oxidized state, and the distance between Fe4′ and Sγ-Cys19 and Sγ-Cys20 is 2.9 Å and 2.7 Å, respectively. As observed for all single cluster variants of MBH, the Fe3 was found in a double conformation. Approximately 75% resided in the native Fe3 position and formed a covalent bond to S4 (Fe3–S4 distance 2.4 Å), consistent with the S4 occupancy. The remaining 25% of the Fe3 were shifted by 1.1 Å toward a more cubic conformation (Fe3′). However, a covalent binding to Sγ-Cys19 was not established (Fe3′ – Sγ-Cys19 distance 3.2 Å), resulting in a three-fold ligation of Fe3′  distance, 2.9 Å; . One exception was found for H2-reduced MBHC19G, which contained a positive mFo-DFc electron density between nickel and iron , nickel-coordinating cysteine (Cys597). Such modification was less evident in the structure of H2-reduced MBHC19G (occupancy below 10%) and can therefore be considered as partly reversible. Notably, we found no evidence of oxygenation at the coordinating cysteine thiolates or of further damage in any of the other crystal structures.The active site structures of the MBH variants carrying modified proximal iron–sulfur clusters were found in the same conformations as previously described for native MBH Fig. S7.14,17 Th; Fig. S7, which ig. S7r-B . The redn Fig. S7. We inteet al.9 already described the IR/EPR-spectroscopic characterization of the MBHC19G/C120G variant, and we will refer to these data mainly in the discussion.To investigate changes in the redox properties for the [NiFe]-active site and the proximal Fe–S cluster of MBH caused by the C19G and C120G exchanges, we carried out IR and EPR spectroscopic experiments in solution, complemented by resonance Raman data obtained from single crystals. Goris C19G displayed a similar band pattern for the [NiFe]-active site as observed for native MBH  correspond to the magnetically coupled active site in the Nir-B state  and Fe3′ (unbound to Cys120) of the proximal cluster in the oxidized MBHC19G crystal structure  and the paramagnetic Nia-C state (∼10%). The spectrum of the reduced MBHC19G variant showed only traces of these reduced states, suggesting different steady state equilibria caused by the modified proximal cluster.The IR spectrum of the reduced MBH variant  was domi Table S3. In addible S3−1 . This ob2-treated MBHC19G was mainly EPR-silent. Only trace amounts of Nia-L were observed. In contrast, H2-reduced native MBH showed a distinct signal typical for the reduced proximal [4Fe–3S] cluster in O2-tolerant MBHs from different organisms , which was assigned to the oxidized medial [3Fe–4S]+ cluster. A similar observation was obtained for native MBH  in the resonance Raman spectrum of H2-reduced MBHC19G crystals when compared to the corresponding spectra of native MBH and MBHC120G  was almost as large as that of Nir-B (∼55%) as revealed by IR spectroscopy catalysts immobilized onto conducting surfaces such as pyrolytic graphite electrodes or metal (oxides) with biocompatible coatings.31–34 The measured currents are proportional to enzymatic turnover making the technique particularly useful for testing the influence of inhibitory gases such as O2 during H2 activation. Here, it is important that the gas composition is tightly controlled during the time the catalytic current is monitored, since changes in activity in response to a particular gas may be slow. The electrochemical cell is thus sealed, and equipped to allow a controlled mixture of gases to flow through the headspace above the solution while the electrode is rotated. Gases to be tested are introduced or removed by adjusting a mass flow controller that rapidly mixes the different components in a precise manner .35 PFE has already been employed to investigate the MBHC19G/C120G variant on a bare pyrolytic graphite electrode.9 The protein rapidly lost its H2-oxidizing activity as soon as O2 was added to the assay solution, which was in deep contrast to the observations for native MBH.9 Here, we tested the capabilities of the MBHC19G and MBHC120G proteins to sustain H2 conversion in the prolonged presence of O2. To avoid irreversible ‘loss’ of protein film from the electrode, seen as a continuous loss of current over time,9 film stability can be improved by covalently attaching the enzyme onto pyrolytic graphite electrodes coated with pyrene-butyric acid-modified multi-walled carbon nanotubes (MWNT).36 The activity of MBHC19G and MBHC120G was followed by cyclic voltammetry (CV) in the presence of 80% H2 (606 μM) under otherwise anoxic conditions  and extended (600 s) low potential pulses (−0.659 V). Nevertheless, the current increase was rather small compared to the recovery observed after O2 removal (Protein film electrochemistry (PFE) is an established method to investigate the H Fig. S12. Both va Fig. S12, promptieriments . The potent drop . The reseadspace .39 Stepwtive MBH . However removal .C19G variant showed a more pronounced response toward O2 exposure. Addition of just 1% O2 (11 μM) resulted in a continuous drop in H2 oxidation current  were observed in all variants. The amount of the Niia-S state (CO peak at 1930 cm−1) seemed to increase continuously during the chronoamperometric measurements, irrespectively of the gas composition. As the quantity of immobilized hydrogenase did not change during the course of the spectro-electrochemical measurements , which accumulates in the presence of the H2/O2 mixture, disappeared completely in native MBH after removal of oxygen. In contrast, the same treatment did not result in the complete elimination of the Nir-B signal for the MBHC120G variant and especially for the MBHC19G variant  spectroscopy under electrochemical control . The MBHtentials  and S13† variant  and S13† variant  and S13†2-mediated methylene blue reduction capacity (under anoxic conditions) were only about 10% of the corresponding values for the native protein. Despite the low yield, we were able to solve the crystal structures of the MBHC120G variant of the oxidized and H2-reduced forms. Among the MBH variants, the MBHC120G protein showed the highest structural plasticity with at least two different states of the proximal Fe–S cluster for each of the two redox conditions. In approximately 50% of the H2-reduced MBHC120G crystals, this new cluster resembles the structure of the reduced native [4Fe–3S] cluster, except that the Cys120 thiolate is partially replaced by an exogenous sulfur species (S4) that coordinates Fe3, resulting in a [4Fe–4S] cluster in a non-cubic form. The other 50% does not contain the S4, making Fe3 part of a cubane, one corner of which is occupied by the Cys19-derived sulfur. To our knowledge, such a novel cubic cluster in which one of the inorganic sulfides is replaced by a cysteine sulfur remains unprecedented. The replacement of Cys120 results in a double conformation of Fe3 that correlates with the exo-sulfur species in terms of the occupancy. The proximal Cys5[4Fe–4S] cluster in oxidized MBHC120G is even more flexible, forming a new cluster scaffold with altered redox properties and electronic structure. In one fraction of the protein, Fe4 is bound to the backbone nitrogen of Cys20 (Fe4′ position), as observed for the native super-oxidized [4Fe–3S] cluster. The other fraction (Fe4) remains in the equivalent position to that found for the reduced state  resulted in a certain redox-dependent flexibility of Fe3, which is comparable but not identical to the situation in MBHC120G. In H2-reduced MBHC19G crystals, a major fraction of Fe3 is not covalently bound to Sγ-Cys120, which is in contrast to the reduced [4Fe–3S] cluster of native MBH. This result can be explained by the fact that sulfide (S4) is a more tightly bound ligand than a thiolate, which leads to coordinative bonding to the shifted Fe3′. The corresponding cubic conformation increases the stability of the cluster. As expected, the Fe4 shift does not occur in the oxidized [4Fe–4S] cluster of MBHC19G. Here, most of Fe3 is covalently bound to Cys120 and not to S4, which is similar to the cluster structure of the C19G variant of E. coli Hyd-1 resulting in a less negative reduction potential.41 Overall, our data show that the covalent bond between Cys120 and Fe3 is crucial for stabilizing the super-oxidized state and thus for the physiological function of the proximal cluster.The crystal structures of HC19G/C120G protein, which contains a four Cys-coordinated [4Fe–4S] species, oxidation resulted in cluster flexibility, indicating that the binding pocket is optimized for an Fe–S cluster of larger dimensions, i.e., the Cys6[4Fe–3S] cluster  of the MBH small subunit. The [4Fe–4S] cluster as present in the H2-reduced MBHC19G/C120G may represent this maturation intermediate, as this species is the most stable among the cluster variants. However, this hypothesis may be supported by future studies on MBH variants in which the canonical cysteine residues were exchanged. We also hypothesize that cluster rearrangement is subsequently triggered by one of the two additional cysteines, Cys19 or Cys120. In the first scenario, Cys19 would initiate the rearrangement, and its thiolate takes over the coordination of Fe1 and Fe4, replacing sulfide S4. The released S4 could either be delivered to the solvent  or swings over towards the position of the thiol group of Cys120. Either of these steps would be accompanied by a shift of Fe3 from the corner position of the cubane. These transformation steps would result in the Cys5[4Fe–4S] cluster as present in the reduced MBHC120G variant. In the second scenario, the Cys120 would first covalently bind Fe3, which should lead to a non-tetrahedral coordination of S4 (represented by the oxidized Cys5[4Fe–4S] cluster of MBHC19G) and its subsequent release. Both scenarios would eventually lead to covalent binding of Cys19 and Cys120 to the corresponding iron ions of the mature Cys6[4Fe–3S] cluster . In contrast to the corresponding spectrum of the native MBH, the proximal cluster of the oxidized MBHC120G remains diamagnetic, as no additional signals and no splitting or broadening of the signals for Nir-B and the medial cluster were observed. The same observation has been made for the oxidized C19G/C120G exchange variants of MBH and E. coli Hyd-1.9,30 In addition, H2-reduced MBHC19G/C120G and Hyd-1C19G/C120G, were EPR-silent, suggesting a fully reduced active site and a diamagnetic medial [3Fe–4Fe]0 cluster. This observation, in turn, is consistent with the EPR spectrum of reduced MBHC120G, which contained only very weak signals interpretable as reduced Fe–S cluster species. The spectrum is also consistent with the predominant formation of EPR-silent, fully reduced Nia-SR sub-states as observed by IR. Although MBHC120G, MBHC19G/C120G, and Hyd-1C19G/C120G showed catalytic activity, their proximal cluster remains non-detectable by EPR after reduction, possibly caused by the presence of an integer spin or an unusual non-integer high spin configuration that is not detectable under the applied measurement conditions. We conclude that the Cys5[4Fe–4S] cluster of MBHC120G and most likely also the proximal Cys4[4Fe–4S] clusters of MBHC19G/C120G and Hyd-1C19G/C120G perform only one low-potential electron transition.15,17,45 This is in accordance with our protein tunneling calculations of the corresponding crystal structures that the exchange of Cys120 with a smaller Gly residue increases the solvent accessibility of the proximal Fe–S cluster in MBHC120G and MBHC19G/C120G crystals .30 This result agrees with our observations for the proximal cluster of the MBHC19G variant, which also seems to allow only the high-potential redox transition.In contrast, a major fraction of the Cys2 incubation, with native MBH being least and MBHC19G most affected. In the case of MBHC19G and partially also for MBHC120G, O2 exposure led to the accumulation of inactive enzyme species that could in part be reactivated by applying a very negative electrode potential. Our PFE results are in line with those obtained for E. coli Hyd-1 variants carrying equivalent exchanges of the supernumerary cysteines.40 Accompanying spectro-electrochemical SEIRA studies indicate that the current decrease upon O2 incubation is related to the accumulation of two inactive states of the catalytic center in all MBH variants. The first one is the inactive Niia-S state, the amount of which increased steadily during the experiment, regardless of which gas mixture was present, indicating no direct correlation between Niia-S state accumulation and O2-induced inactivation. Besides Niia-S (and the reduced states), all three investigated MBH variants formed the Nir-B state during H2/H+-cycling in the presence of O2. The fact that for the MBHC19G protein in the Nir-B state, current recovery occurred mainly after applying negative potential pulses indicates major kinetic limitations regarding its reactivation ability, which resulted in only a marginal current recovery upon O2 removal. In fact, the redox potential of the Nir-B state of the C. necator MBH lies at −110 mV.47 Reactivation of the Nir-B state requires one proton and one electron  leads to the formation of the super-oxidized state of the [4Fe–3S] cluster, as suggested previously45 and supported by the fact that O2-mediated full oxidation to the super-oxidized state exclusively takes place if the MBH is still coupled to the cellular quinone pool , however, results in the movement of Fe4 back into the Fe–S cluster core. As this reorganization is time-consuming,45 the [4Fe–3S]5+ cluster functions as an implemented time-delay preventing premature reactivation of the Nir-B state in the case of H2 scarcity under otherwise oxic conditions. Importantly, the active site protection mechanism described above does not preclude the previously described model of O2 detoxification by four electrons and four protons delivered to the active site in the reverse direction to reduce O2 to harmless water.9,14,18,19,37,48 In the proposed new mechanism, activity can be switched off under electron-deficient conditions even before O2 would gain access to the catalytic center. In fact, Knallgas bacteria such as C. necator usually face an (micro)aerobic environment in which H2 occurs transiently and only in traces.4Removal of the active site OH ligand under H9 on plasmid pCH1229.37 The modified plasmids were digested with SpeI/SmaI and the resulting 1171 bp fragments were transferred to SpeI/SmaI-cut pJH2430 (Litmus28 containing PMBH-hoxKStrep-hoxV) resulting in plasmids pJH5116 (C19G) and pJH5127 (C120G). Plasmids pJH5116 and pJH5127 were digested with SpeI/BstZ17I and the resulting 2581 bp fragments were ligated into SpeI/BstZ17I-cut pCH785 (ref. E. coli S17-1 (ref. C. necator HF210 resulting in strains HP83 (C19G) and HP84 (C120G).The amino acid exchanges C19G and C120G in the small MBH subunit HoxK were introduced by the QuikChange (Agilent) method using primers #793/#794 for C19G and #795/#796 for C120G785 ref.  resultin785 ref.  resultin9 with SpeI/BstZ17I and ligating the resulting 2581 bp fragment into SpeI/BstZ17I-cut pCH785 resulting in plasmid pJH4052. The latter was digested with SpeI/XbaI and the resulting 21.5 kbp fragment was ligated into XbaI-cut pEDY309 resulting in plasmid pJH4081 which was conjugated into C. necator HF210 resulting in strain HP29. Recombinant C. necator strains were cultivated and the MBH variants purified as described elsewhere.51The double amino acid exchange C19G/C120G in the small MBH subunit HoxK was introduced by digesting pCH1487 C19G, MBHC120G and MBHC19G/C120G variants were crystallized under either aerobic (oxidizing) or anaerobic, reducing conditions (in the presence of H2) as described previously.14,17 In brief, the individual MBH variants were crystallized by applying the sitting-drop vapor diffusion method in 24-well Linbro plates (Jena Bioscience) at a concentration of 10 mg mL−1 at 277 K and 283 K for oxidized and reduced crystals, respectively. The reservoir solution contained 20–30% polyethylene glycol 3350, 100 mM bis-(2-hydroxy-ethyl)-amino-tris(hydroxymethyl)-methane buffer at pH 5.5–6.5. The drop ratio of precipitant and protein solution varied between 0.5 and 2. Brownish crystals appeared within 2–4 days and upon collection were directly flash-frozen in liquid nitrogen (LN2).The purified MBH2-reduced MBHC19G/C120G was collected at BESSY II, beamline BL14.1 (ref. 8POV). For oxidized MBHC19G/C120G, data collection was executed at ESRF, beamline ID29 (ref. 8POU). For anomalous data collection, the wavelength was shifted towards 1.734 Å. The best diffracting data for oxidized and H2-reduced MBHC19G/C120G were collected at ESRF, beamline ID29 (ref. 8POW and 8POX). Data for oxidized and H2-reduced MBHC120G crystals were collected at the ESRF, beamline ID29 (ref. 8POY and 8POZ). Additionally, anomalous data at 1.734 Å was recorded for MBHC120G. Images were indexed, integrated and scaled using the XDS program package54 and the CCP4 program SCALA.55,56 All crystals showed the orthorhombic space group P212121. Table S22-reduced native (reduced) MBH (PDB entry 3RGW14) and as-isolated (super-oxidized) native MBH (PDB entry 4IUB17) as the initial search models for the MBH variants. Molecular replacement was achieved using the CCP4 program PHASER.57 Phasing and heavy atom search for analyzing anomalous data were performed by using the SHELX package.58 Statistics for anomalous crystallographic data collection are provided in Table S2.PHENIX and CCP4 program REFMAC5 using refinement strategies  and simulated-annealing  to overcome potential phase biases.55,59–61 First water molecules were depicted with the automatic search method in PHENIX.60 Manual model building and electron density interpretation were performed in COOT.62 After every round of manual model building, energy minimization and B-factor refinement were performed with REFMAC5.61,63 The final crystal structures were validated using the RCSB PDB Validation Server,64MolProbity server65 and WHAT IF server.66 Solvent accessibility was calculated in PyMOL using the program CAVER3.0.67 All molecular graphics representations were created in PyMOL.68Diffraction data were collected at the synchrotron facilities BESSY II  and ESRF . The best diffracting data for H4.1 ref.  with a RD29 ref.  with a PD29 ref.  with a PD29 ref.  with a P2), the electrode was first polished for 30 s with an aqueous slurry of α-alumina  and sonicated for 5 s in purified water, before MBH variant solution  was repeatedly applied and withdrawn from the electrode surface over a period of 10–30 s. The electrode was then rinsed in purified water and used immediately.To prepare each film of non-covalently bound enzyme on pyrolytic graphite ‘edge’ (PGE) disk electrodes  in DMF (16 μL of 1 mg mL−1) was placed on the surface of a PGE-disk electrode and allowed to dry overnight. The MWNT-coated PGE electrodes (geometric area 0.2–0.28 cm2) were then coated with a solution of 1-pyrenebutyric acid (Py) in DMF (10 mM) for 1 h to obtain strong π–π stacked MWNT-Py assemblies on PGE (PGE/MWNT/Py). The electrodes were rinsed in purified water and the free carboxylic groups of Py were activated for 20–40 min using a freshly prepared solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide  + N-hydroxy sulfosuccinimide  before treating with MBH variant solutions at room temperature for 60 min to make covalent attachments via the surface lysine residues (∼33 for MBH from C. necator). This procedure resulted in modified electrodes denoted PGE/MWNT/Py-MBH. The electrodes were then rinsed in purified water and used immediately or stored at room temperature in 50 mM potassium phosphate, 100 mM NaCl buffer, pH 5.5.Covalent immobilization of MBH on electrodes was performed as described previously.2 atmosphere (O2 <2 ppm). The enzyme-coated electrodes were placed in enzyme-free buffered electrolyte  so that all enzyme molecules addressed in the experiments were subjected to the same regime of strict potential control. In all experiments the electrode was rotated at a constant rate (>2500 rpm) to provide efficient supply of substrate and removal of product. The electrode rotator (EcoChemie) was placed in a specially designed, gas-tight, glass electrochemical cell incorporating a Pt wire counter electrode. A saturated calomel reference electrode (SCE) was contained in a sidearm containing 100 mM NaCl, separated from the main cell compartment by a Luggin capillary. Potentials (E) are quoted with respect to the standard hydrogen electrode (SHE) using the conversion ESHE = ESCE + 0.241 V at 25 °C.70Protein film electrochemistry experiments were carried out using an electrochemical analyzer (Autolab PGSTAT128N) controlled by a PC operating NOVA software (EcoChemie) in an anaerobic glovebox (M. Braun) comprising a N2 , Ar, and O2 (BOC gases) created using mass flow controllers (Sierra Instruments).Experiments were performed in phosphate buffer  containing NaCl  and titrated to the desired pH at the experimental temperature. Experiments were performed under gas mixtures of H2, 95% (v/v) N2) and incubated under a stream of humidified H2 for 45 min. IR transmission cells and EPR tubes were purged with H2 prior to protein transfer to avoid re-oxidation by residual oxygen. For re-oxidation, reduced samples were exposed for 60 min to a stream of humidified air. EPR samples were transferred into quartz EPR tubes, flash-frozen in cold ethanol (210 K) and stored in liquid N2 (77 K) for further analysis.For measurement of oxidized MBH variants, as-purified protein samples were concentrated with an Amicon Ultra concentrator to approx. 0.2 mM. For reduction, as-purified MBH samples were transferred into an anaerobic glovebox containing a forming gas atmosphere (5% (v/v) HAll IR transmission spectra were measured at 10 °C in a gastight cell with a sandwich configuration  using a Bruker Tensor 27 FTIR spectrometer equipped with a liquid nitrogen cooled MCT detector and controlled by the Bruker OPUS 7.0 software. The sample compartment was purged with dry air. Absorbance spectra of the sample were calculated using single-channel spectra of the pure buffer as reference.All EPR measurements were carried out on a Bruker EMXplus spectrometer equipped with an ER 4122 SHQE resonator, an Oxford EPR 900 helium flow cryostat and an Oxford ITC4 temperature controller. Baseline correction was carried out by subtracting a buffer spectrum measured under the same conditions. Spectra were further corrected by using a polynomic or spline function based on broad spectral regions where no signal occurs. Experimental parameters: microwave power: 1 mW; microwave frequency: 9.29 GHz; modulation amplitude: 10 G; modulation frequency: 100 kHz.2. Experiments were performed at 80 K using a liquid-N2-cooled cryo-stage (Linkam Scientific instruments) for temperature control. The Ar+ ion laser with an excitation wavelength of 458 nm was focused at a 2–4 μm spot on the surface of the single crystal with a beam power of 1–2 mW. For frequency calibration of the spectra, toluene was used as an external standard.Resonance Raman spectra were measured on a LabRam HR-800 Jobin Yvon confocal Raman spectrometer connected to a charge-coupled device (CCD) cooled with liquid-N−1 with a spectral resolution of 4 cm−1 on a Bruker IFS 66 v/S spectrometer equipped with a photovoltaic MCT detector. For a single spectrum, 400 scans were added up.71 For the protein immobilization, biocompatible surfaces were created by immersing Au electrodes for 12 h in an ethanolic solution of 6-mercaptohexanoic acid and 6-mercapto-1-hexanol in a 1 : 9 ratio, with initial concentrations of 1 mM. Thereby, a mixed self-assembled monolayer (SAM) was formed on the electrode surface. Subsequently, the MBH variants were immobilized according to ref. 2), a Pt mesh as a counter electrode, and an Ag/AgCl 3 M KCl  reference electrode. A schematic representation of the homemade SEIRA spectro-electrochemical cell used in this work is depicted elsewhere.72 Electrochemical control was achieved using a Metrohm μAutolab potentiostat.SEIRA spectroscopic measurements were performed in the Kretschmann-ATR configuration using a Si prism under an angle of incidence of 60°. On the flat surface of the prism thin, nanostructured Au films were formed by electroless  deposition. The corresponding SEIRA spectra were recorded from 4000 to 1000 cmAll experimental data and crystallographic analyses are available in the ESIP. S., O. L and I. Z. conceived and supervised the project. J. F. and O. L. constructed the MBH variants. S. F. and J. F. purified the MBH samples and analyzed them biochemically. A. S., J. K., S. F., J. F., and P. S. managed the MBH crystallization. A. S., J. K., and P. S. collected the X-ray and anomalous diffraction data, performed data processing and solved the MBH structures. A. S., J. K. and P. S. refined and interpreted the MBH structures. C. L. recorded and analyzed EPR data supported by C. T. IR and RR and spectro-electrochemistry data were obtained and analyzed by S. K., C. L. and I. Z. R. M. E. and F. A. A. recorded and analyzed electrochemistry data; A. S., J. K., S. F., C. L., S. K., I. Z., O. L. and P. S. wrote the paper with contributions from all co-authors.patrick.scheerer@charite.de), O. L. (oliver.lenz@tu-berlin.de), and I. Z. (ingo.zebger@tu-berlin.de).The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P. S. (SC-014-D3SC03739H-s001


This trial evaluated the safety and efficacy of ipatasertib in combination with carboplatin, carboplatin/paclitaxel, or capecitabine/atezolizumab in patients with metastatic triple–negative breast cancer (mTNBC).Eligibility criteria were mTNBC, RECIST 1.1 measurable disease, no prior use of platinum for metastatic disease (Arms A and B), and no prior exposure to immune checkpoint inhibitor (Arm C). Primary endpoints were safety and RP2D. Secondary endpoints were progression–free survival (PFS), response rate, and overall survival.n = 10) was ipatasertib 300 mg daily, carboplatin AUC2, and paclitaxel 80 mg m−2 days 1, 8, and 15 every 28 days. RP2D for Arm B (n = 12) was ipatasertib 400 mg daily and carboplatin AUC2 days 1, 8, and 15 every 28 days. RP2D for Arm C (n = 6) was likely ipatasertib 300 mg 21 days on 7 days off, capecitabine 750 mg m−2, twice a day, 7 days on 7 days off, and atezolizumab 840 mg days 1 and 15 every 28 days. The most common (≥10%) grade 3-4 AEs at RP2D for Arm A (N = 7 at RP2D) were neutropenia (29%), diarrhea (14%), oral mucositis (14%), and neuropathy (14%); Arm B had diarrhea (17%) and lymphopenia (25%); and Arm C had anemia, fatigue, cognitive disturbance, and maculopapular rash (17% each). Overall responses at RP2D were 29% Arm A, 25% Arm B, and 33% Arm C. PFS was 4.8, 3.9, and 8.2 months for patients on Arms A, B, and C, respectively.RP2D for Arm A . This article evaluates the safety and efficacy of ipatasertib in combination with carboplatin, carboplatin/paclitaxel, or capecitabine/atezolizumab in patients with metastatic TNBC. Ipatasertib is a selective pan-AKT inhibitor and displays synergy with chemotherapy preclinically. This study was designed to determine the dose, safety, and preliminary efficacy of ipatasertib plus carboplatin, carboplatin/paclitaxel, or capecitabine/atezolizumab in mTNBC. The combination was safe and well-tolerated at the recommended doses, with empirically higher efficacy seen in tumors harboring PIK3CA/AKT/PTEN alterations. In addition, androgen receptor-positivity was associated with an empirically higher response rate. This study was stopped early due to a Phase III study combining ipatasertib plus paclitaxel that failed to show PFS or OS benefit. Further analysis is warranted in understanding the role of AKT inhibition in the treatment of TNBCs.1 A number of novel agents have recently been granted FDA approval, including PARP inhibitors for germline BRCA mutated tumors,3 immune checkpoint inhibitor (ICI) for programmed death-ligand 1 (PD-L1) positive TNBC,4-6 and antibody drug conjugates targeting the Trop-2 receptor.8 Despite these advances, the median overall survival (OS) of patients with mTNBC remains limited at approximately 18 months.9 In the ASCENT trial, the median OS was 12.1 months  with sacituzumab govitecan as 3+ lines of therapy, and 6.7 months  with chemotherapy .8 Therefore, more effective therapeutic strategies targeting underlying genomic drivers, as well as novel combinations are urgently needed.Metastatic triple-negative breast cancer (mTNBC), defined as lack of estrogen receptor (ER), progression receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression, remains a disease of unmet need. This is largely attributed to tumor heterogeneity and chemotherapy resistance.11 The mTOR inhibitor everolimus in combination with eribulin or carboplatin has shown moderate activity.13 Ipatasertib is a selective ATP–competitive small–molecule inhibitor of AKT that preferentially targets active phosphorylated Akt (pAkt) and is potent in cell lines with evidence of Akt activation. Ipatasertib displays synergy when combined with taxanes or other chemotherapeutic agents  in vitro.12-14 In the LOTUS trial, combination of paclitaxel 80 mg m−2  and ipatasertib 400 mg po days 1-21 every 28 days showed modestly improved progression–free survival (PFS). More pronounced improvement was seen in tumors with alteration of the PIK3CA/AKT/PTEN pathway.15 The combination regimen was well-tolerated with 23% of grade ≥3 diarrhea and 18% grade 3 neutropenia. Phase III IPATunity trial failed to confirm the efficacy of adding ipatasertib to paclitaxel in both TNBC (Cohort A)16 and ER+ MBC as first–line therapy (Cohort B),17 which is intriguing. This controversial result may indicate a different chemotherapy backbone is needed for optimized therapeutic effect.The Cancer Genome Atlas data demonstrated that the most frequently altered genomic drivers in TNBC include the phosphatidylinositol 3-kinase-AKT-mTOR (PI3K-AKT-mTOR) signaling pathway.P = .66).18 However, carboplatin showed higher ORR and longer PFS in comparison with docetaxel in patients with gBRCA+ mTNBC: ORR 68% vs 33% (P = .01) and PFS 3.1 months  vs 4.4 months .19 A high proportion of TNBC tumors exhibit BRCAness, which indicate these tumors are highly sensitive to platinum agents.21 A weekly carboplatin–paclitaxel regimen has been tested in multiple clinical trials and has shown efficacy and acceptable safety profiles.22 Weekly paclitaxel (100 mg m−2) was combined with weekly carboplatin AUC 2, days 1, 8, and 15 on a 4–week schedule in a cohort of advanced breast cancer patients. The regimen was effective with a response rate (RR) of 62% and was well tolerated.23 We previously found that in the patient–derived xenograft model of mTNBC, carboplatin and ipatasertib were synergistic in tumor suppression.24 Based on this evidence, we hypothesize that carboplatin or carboplatin/paclitaxel in combination with ipatasertib would have a synergistic effect in mTNBC. Hence Arms A and B of this clinical trial were designed to determine the dose and safety of ipatasertib plus carboplatin or carboplatin/paclitaxel to obtain initial evidence of efficacy. A continuous dosing schedule of ipatasertib was used in both Arm A and Arm B.DNA–damaging agents such as platinum drugs are active in TNBC. The randomized Triple Negative Breast Cancer Trial (TNT) showed no overall response rate (ORR) difference between carboplatin and docetaxel in the overall population: 31.4% vs 34%  with mTNBCs. Interestingly, responses were high irrespective of PD-L1 status or PI3K/AKT/PTEN alteration status.31 Based on this evidence, the aim of Arm C was to test the safety and preliminary efficacy of the triplet combination of ipatasertib (intermittent dosing), capecitabine, and atezolizumab in patients with mTNBC.The Impassion130 study demonstrated the addition of atezolizumab (anti–PD-L1) to This open-label single institutional Phase I/Ib trial was conducted between May 2019 and June 2022 with institutional review board (IRB 18496) approval at the City of Hope National Cancer Center. The trial was conducted in accordance with the World Medical Association Declaration of Helsinki, International Conference on Harmonization Good Clinical Practice guidelines, and the US code of federal regulations. Informed consent forms were signed by all patients prior to study entry. This study is registered at the ClinicalTrials.gov under number NCT03853707.Main eligibility criteria were: ≥18 years; histologically confirmed TNBC defined by ER or PR ≤ 10% by IHC and HER2 negative per ASCO/CAP guidelines; ECOG performance status 0-1; life expectancy ≥3 months; RECIST 1.1 measurable disease for Arm C only (patients on Arms A and B could have non–measurable disease), available baseline archival tissue for PIK3CA/AKT/mTOR status, and adequate organ function. Main exclusion criteria included unresolved grade 3 toxicities, prior exposure to PIK3CA/AKT/mTOR pathway inhibitors, prior exposure to carboplatin (Arms A and B) for metastatic disease, prior exposure to paclitaxel (Arm A) for metastatic disease, prior exposure to capecitabine or ICIs (Arm C) in the metastatic setting, or untreated or unstable brain metastasis or leptomeningeal metastasis.The primary objective of the study was to evaluate safety and determine the recommended Phase II dose (RP2D) of ipatasertib plus carboplatin and paclitaxel (Arm A), carboplatin (Arm B), or capecitabine and atezolizumab (Arm C). Secondary endpoints were RR, PFS, and OS. Responses were assessed by RECIST 1.1, and safety analysis was carried out based on toxicities assessed by CTCAE 5.0. Immune-related adverse events (irAEs) were also collected for Arm C.Patients underwent tumor assessments with CT scan of chest, abdomen, pelvis, and bone scan of at baseline, every 3 cycles (12 weeks) following treatment initiation regardless of dose delays, until radiographic disease progression per RECIST v1.1 or intolerance. All measurable and evaluable lesions were re-assessed at each subsequent tumor evaluation. An objective response was confirmed by repeat assessments ≥4 weeks after initial response.32 and Phase II LOTUS studies as: ipatasertib 400 mg po daily 3 weeks on/1 week off and paclitaxel 80 mg m−2 days 1, 8, and 15 of every 4-week cycle.33The combination of paclitaxel and ipatasertib was determined to be safe and well tolerated in previous Phase Ib (PAM 4983g)−2 IV on days 1, 8, and 15 of every 28 days. For Arm B, ipatasertib was administered at a starting dose level of 400 mg oral daily for 28 days plus carboplatin AUC 2 on days 1, 8, and 15 every 28 days. For Arm C, ipatasertib was administered at a starting dose level of 300 mg oral daily for 21 days on and 7 days off, capecitabine at a dose of 750 mg m−2 7 days on and 7 days off, and atezolizumab at a dose of 840 mg IV on days 1 and 15 every 28 days. For Arms A and B, continuous dosing of ipatasertib was used. For Arm C, ipatasertib was used for 21 days on and 7 days off. For the Phase I/Ib design, the Phase I Queue (IQ) 3 + 3 design was used.34 This method has been shown to reduce study duration by an average of approximately 23% for a typical Phase I study, with a median increase in the number of patients of 1-4, while reducing the number of patients turned away due to lack of slots.Treatment Arms are shown in To confirm the RP2D obtained from the dose escalation, and for an initial assessment of response, correlatives, and PFS estimates, an additional cohort of patients was enrolled until the number treated and evaluable for DLT considerations at the recommended Phase II dose was 14 patients for Arm B and Arm A. All eligible patients who started treatment at the recommended Phase II dose were considered in the calculation of the RR.Arms A and B had an expected sample size of 14 patients, for an expected total sample-size of 28 patients. Protocol allowed for the addition of cohorts (6-8 patients) if level 1 was not well-tolerated (a lower dose was necessary).For Arm C safety-lead in, a 3-at-risk design was utilized to assess toxicity for the combination therapy. The DLT period was 1-cycle (28 days). Each participant remained on the dosing level according to the escalation dose level they were enrolled in, and intra-dose level escalations were not allowed, even if the MTD was defined at a higher dose level. If a patient came off study in the first 28 days for any reason outside of toxicity , this patient was not considered as evaluable for DLT and this patient was replaced.When a maximum tolerable dose (MTD) level was defined by the dose escalation portion of the study, and the recommended Phase II dose (RP2D not to exceed the MTD) was selected, additional patients were accrued to confirm the tolerability of the regimen. The plan was for at least 12 patients to be treated at the RP2D to confirm tolerability. Additional patients beyond the 12 at the RP2D could be accrued if the total number of patients accrued did not exceed 21 patients. With 12 patients, any specific severe toxicity with 20% incidence would be observed with 93% probability.Survival endpoints were evaluated using Kaplan-Meier methods. Clinical activity was described based on the secondary objectives, with a description of the activity based on PD-L1 status. Other biological correlative studies were considered exploratory in the context of this limited Phase I study.35 Genomic sequencing was performed using Tempus xT version 4 assay consisting of 648 genes with single nucleotide variants, indel, and translocations measured by next-generation sequencing.36 The copy number gain limit of detection was set as 30% and copy number loss at 40% tumor purity. Tumor mutational burden was reported as the quantity of somatic single nucleotide variations (SNVs) and indels (including benign) measured as mutations per million coding base pairs. PD-L1 was reported through the commercial testing platform Tempus using SP142 antibody. PD-L1 positive was defined by at least ≥1% on immune cells.Tumor biopsies were formalin-fixed paraffin-embedded (FFPE). Percentage of stromal tumor infiltrating lymphocytes (TILs) in tumor was evaluated using H&E diagnostic sections per International Immuno-Oncology Biomarker Working Group on Breast Cancer Guidelines.16 Baseline patient characteristics are listed in A total of 28 patients with metastatic TNBC were enrolled between May 2019 and April 2021: 10, 12, and 6, respectively, to Arms A, B, and C . The stu−2, days 1, 8, and 15, every 28 days. Grade 3 AEs at RP2D were: 2/7 (29%) leukopenia, 2/7 (29%) lymphopenia, 2/7 (29%) neutropenia, and 1/7 (14%) each of diarrhea, oral mucositis, and peripheral sensory neuropathy. One patient in Arm A had both grade 3 oral mucositis and grade 3 peripheral sensory neuropathy  leading to dose delay (less than 75% planned ipatasertib dosing) . Due to uropathy . Of a touropathy . Arm C guropathy .As previously mentioned, of the 10 patients in Arm A, 3 had DLTs including 2 patients with grade 2-3 diarrhea and 1 patient with grade 3 stomach pain within the first 28-day cycle, which led to de–escalation to dose -1 with ipatasertib (300 mg daily). Of the 12 patients in Arm B, 1 had grade 3 maculopapular rash in cycle 1, which led to dose delay of ipatasertib (<75% completion) during the first 28 days. Of the 6 patients in Arm C, 1 had grade 3 maculopapular rash attributed to atezolizumab. After appropriate treatment, both patients with maculopapular rash had resolution of rash .−2 . The RP2D for Arm B was ipatasertib 400 mg daily/carboplatin AUC2 . The likely RP2D for Arm C was ipatasertib 300 mg , capecitabine 750 mg m−2 bid (7 days on 7 days off), and atezolizumab 840 mg days 1 and 15 every 28 days, with 1 DLT in 6 patients .Median (range) of cycles completed was 4 (2-15), including 4 (2-7) for Arm A, 4 (2-15) for Arm B, and 6 (2-10) for Arm C. A total of 11 (39%) had dose delay, including 4 (40%) in Arm A, 6 (50%) in Arm B, and 1 (17%) in Arm C. Ten (36%) had dose reduction, including 6 (60%) in Arm A, 3 (25%) in Arm B, and 1 (17%) in Arm C. The RP2D for Arm A was ipatasertib 300 mg daily, carboplatin AUC2 and paclitaxel 80 mg mN = 28), the overall responses were: 1 (4%) CR, 6 (21%) PR, 13 (46%) SD, 7 (25%) PD, and 1 (4%) HER2+ at time of progression. A total of 10 patients had clinical benefit rate (CBR = CR + PR + SD) at 6 months without progression, including 2 (20%) in Arm A, 5 (42%) in Arm B, and 3 (50%) in Arm C. Duration of response (CR and PR) was 4 and 7 months for Arm A; 6, 9, and 15 months for Arm B; and 9 and 15 months for Arm C (1 patient was still on treatment). The median follow-up for Arm A was 24 months and Arm C was 20 months. Reverse Kaplan–Meier method could not be used to determine median follow-up for arm B; but was 10 months and 21 months for the 2 patients on follow-up.The best responses for patients in Arm A (carboplatin/paclitaxel plus ipatasertib) included 2 (20%) PR , 4 (40%) SD, and 4 (40%) PD. Arm B (carboplatin plus ipatasertib) best response included 1 (8%) CR, 2 (17%) PR, 6 (50%) SD, and 3 (25%) PD. Arm C  best response included 2 (33%) PR, 3 (50%) SD, and 1 (17%) was called progressive disease, but we noted the patient was HER2+ on biopsy of progressive disease . The oveN = 10) was 11.2 months , Arm B (N = 12) was 17.0 months , and Arm C (N = 6) is NA , Arm B was 3.9 months , and Arm C was 8.2 months . The median OS for Arm A (n = 10), Arm B (n = 12), and Arm C (n = 6), including best response and reason for patient going off treatment (A swimmer plot with response to treatment over time is shown for Arm A (reatment . One patPD-L1 testing for Arm C patients showed 4 patients who were PD-L1 positive by SP142 and 2 patients who were PD-L1 negative . No clean = 10) and 17 were AR negative (1 had no AR results). In the AR positive population, the responses were 1/10 CR, 3/10 PR, 5/10 SD, and 1/10 PD or 4/10 responders .A total of 10 patients were AR positive .NextGen exome sequencing was performed with commercial testing . A total6 The data presented by this study may provide proof of principle for a combination of ipatasertib, capecitabine, and atezolizumab for treatment of mTNBC.The results of the current trial provide evidence that combining ipatasertib with continuous daily dosing with weekly carboplatin or carboplatin/paclitaxel is safe and has modest clinical activity in patients with mTNBC. In addition, ipatasertib (21 days on and 7 days off) combined with capecitabine and atezolizumab is safe and showed clinical activity. In patients with PIK3CA/AKT/PTEN alteration, ORR was 33% . In AR+ TNBC, ORR was 40% (4/10). Due to limited sample size, these results are hypothesis-generating. The KEYNOTE-355 trial already established ICI plus chemotherapy for standard management of first–line patients with PD-L1+ TNBC.P < .0001).4 The Phase III confirmatory trial IMpassion131 trial did not meet the primary end point of PFS benefit as in PD-L1+ mTNBC as first–line therapy  and no OS benefit seen in the intention to treat population. In the KEYNOTE 355 study, patients with PD-L1+ TNBC  pembrolizumab plus chemotherapy had improved PFS compared with chemotherapy alone .37 The precise underlying etiology of efficacy difference observed in these ICIs remains unclear. Capecitabine was previously tested in combination with ICIs. Shah et al. reported an ORR of 26% and CBR of 28% (N = 15) in a cohort of patients with mTNBC treated with capecitabine and pembrolizumab combination.28 Page et al. reported capecitabine and pembrolizumab demonstrated a 12-week ORR of 43% and PFS of 5.6 months.38 In our study, ORR of 40% with ipatasertib, capecitabine, and atezolizumab in PD-L1 unselected patients is significant, despite the limited number of patients.ICI plus chemotherapy combinations are now standard of care for PD-L1 positive mTNBC. Atezolizumab was initially granted FDA approval in March 2019 based on significant PFS benefit when atezolizumab was combined with nab–paclitaxel vs nab–paclitaxel alone seen in from IMpassion130 trial .33 However, the Phase III IPATunity130 cohort A failed to show PFS benefit from the addition of ipatasertib to paclitaxel in patients with mTNBC harboring PIK3CA/AKT1/PTEN mutations.39 Similarly, adding ipatasertib to paclitaxel did not improve efficacy in PIK3CA/AKT1/PTEN–altered HR+ HER2–negative advanced breast cancer.17 In the FAIRLANE trial, adding ipatasertib to 12 weeks of paclitaxel for early TNBC did not clinically or statistically significantly increase the pCR rate, although the ORR by MRI was numerically higher with ipatasertib.40 In the current study, the modest efficacy seen in the total population is consistent with IPATunity130 cohort A. Preclinical studies have shown co–administration of ipatasertib and ICI may enhance efficacy by retaining the stem–like phenotype of memory T cells, thus preventing exhaustion.42 A multi–arm basket-trial with immunotherapy–based treatment is currently ongoing (MORPHEUS TNBC NCT03424005) to further test ipatasertib combinations.43In LOTUS trial, ipatasertib added to first–line paclitaxel for mTNBC improved PFS with an enhanced effect in patients with PIK3CA/AKT1/PTEN–altered tumors.45 Lehmann et al. demonstrated that activating PIK3CA mutations is enriched in LAR TNBC; the growth and viability of androgen receptor (AR) positive TNBC cell line models are significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist.46 LAR TNBC PDX models were significantly enriched in PIK3CA and AKT1 mutations, had higher levels of luminal–androgen–like gene expression, and had a higher PI3K pathway protein activation score than other TNBC subtypes.47 These results provide rationale for AR antagonists in combination with PI3K/AKT/mTOR inhibitors. In the current study, our finding of ORR 44% in AR+ TNBC is encouraging. Confirmatory studies are required to better understand the role of ipatasertib in AR+ TNBC.TNBC is molecularly heterogeneous with at least 4-6 molecular subtypes defined by mRNA expression. Among these, approximately 10% are luminal androgen receptor positive (LAR).P = .06). Median OS was 19.1 months with capivasertib plus paclitaxel and 12.6 months with placebo plus paclitaxel . In patients with PIK3CA/AKT1/PTEN–altered tumors (n = 28), median PFS was 9.3 months with capivasertib plus paclitaxel and 3.7 months with placebo plus paclitaxel .48 The results of the Phase III randomized study with capivasertib and CAPItello-290 are eagerly awaited (NCT03997123).Other AKT inhibitors are currently undergoing vigorous clinical investigation. In the PAKT trial, the addition of the pan-AKT inhibitor capivasertib to first-line paclitaxel therapy for TNBC resulted in longer PFS and OS. The median PFS was 5.9 months with capivasertib plus paclitaxel and 4.2 months with placebo plus paclitaxel (hazard ratio [HR], 0.74; 95% CI, 0.50-1.08; 1-sided There are several limitations of this study including early discontinuation due to withdrawal of funding, limited enrollment per arm, variation of chemotherapy backbone, lack of randomized Phase II design, and underlying molecular heterogeneity of mTNBC. An appropriately designed randomized Phase II trial may facilitate better understanding the role of ipatasertib in treating patients with TNBC who have specific molecular alterations or phenotypes.Results from this study showed that continuous dosing of ipatasertib in combination with carboplatin-based therapy or capecitabine/atezolizumab is safe; however, moderate efficacy was seen in TNBC patients with PI3K/AKT/PTEN alterations. Encouraging efficacy was seen in luminal androgen receptor TNBC. Future studies with larger patient cohorts and randomized designs are required to confirm the current findings.oyad026_suppl_Supplementary_TablesClick here for additional data file.


Seasonal influenza A and B viruses may cause severe infections requiring therapeutic interventions. Baloxavir, the latest antiviral drug approved against those infections, targets the endonuclease activity encoded by the polymerase acidic (PA) protein. While appearing effective at cessation of viral shedding, baloxavir demonstrated a low barrier of resistance. Herein, we aimed to assess the impact of PA-I38T substitution, a major marker of baloxavir-resistance, on the fitness of contemporary influenza B viruses. Recombinant wild-type (WT) influenza B/Phuket/2073/13 (B/Yamagata/16/88-like) and B/Washington/02/19 (B/Victoria/2/87-like) viruses and their respective PA-I38T mutants were used to evaluate replication kinetics in vitro, using A549 and Calu3 cells, and ex vivo, using nasal human airway epithelium (HAE) cells. Infectivity was also assessed in guinea pigs. In the B/Washington/02/19 background, there were no major differences between the recombinant WT virus and its I38T mutant when viral replication kinetics were evaluated in human lung cell lines and HAE as well as in nasal washes of experimentally infected guinea pigs. By contrast, the I38T mutation moderately impacted the B/Phuket/2073/13 viral fitness. In conclusion, contemporary influenza B viruses that may acquire baloxavir-resistance through the PA-I38T substitution could retain a significant level of fitness, highlighting the importance of monitoring the emergence of such variant. Seasonal influenza epidemics are currently caused by influenza A (H1N1 and H3N2) strains, in addition to influenza B viruses belonging to B/Yamagata/16/88 and B/Victoria/2/87 genetic lineages. Of note, strains from the B/Yamagata lineage have not circulated in the past few years .Neuraminidase (NA) inhibitors (NAI), including oseltamivir, zanamivir and peramivir, constitute a valuable complement to annual vaccines for the control of seasonal influenza infections. Clinical benefits provided by NAIs are particularly significant when the therapy is instituted within 48 h after the onset of symptoms . InteresBesides NA, influenza polymerase proteins have also been targeted for the development of anti-influenza compounds . BaloxavWe and others previously investigated the impact of the I38T PA substitution in A(H1N1), A(H3N2) and B strains on in vitro replicative properties and virulence/transmissibility using various animal models  ,18,19,20ST6-GalI-MDCK cells overexpressing the ⍺2-6 sialic acid receptor  (kindly Recombinant influenza B wild-type (WT) viruses and their respective I38T PA mutants were previously generated by reverse genetics using the backbones of Victoria-like   vaccine The in vitro replication kinetics of the recombinant WT and mutant viruses were determined in 12-well plates of confluent A549 or Calu3 cells infected with a multiplicity of infection (MOI) of 0.002. Briefly, cells were washed with PBS , and the infection was performed in DMEM supplemented with 0.5 μg/mL TPCK-treated trypsin  for A549 cells and in MEM supplemented with 1 μg/mL TPCK for Calu3 cells. Supernatants were harvested at 12, 24, 48, 72, 96 and 120 h post-inoculation (p.i.) and titrated by qRT-PCR (see below) or by 50% tissue culture infective dose (TCID50) assays on ST6GalI-MDCK cells with the Reed and Muench endpoint method .2 atmosphere for 30 min. Then, the inoculum was harvested, and the HAE was cultured at the air-liquid interface. Apical washes were collected at 24, 48, 96, and 120 h p.i. and titrated by quantitative RT-PCR (see below) or by TCID50 assays.The apical poles of HAE inserts were gently washed with warm opti-MEM before infection. The epithelium was infected with an MOI of 0.002. Viruses were adsorbed at 33 °C under a 5% CO® 480 system (Roche Molecular System).Viral RNA extraction was performed from 90 μL of supernatants from A549 and Calu3 cells or of HAE apical washes using the MagNA Pure LC system . Reverse transcription quantitative PCR (qRT-PCR) assay was performed using primers targeting the influenza NS1 gene of influenza B viruses (available upon request). This assay was performed with the QuantiTect Virus + ROX Vial Kit  on a LightCycler5 PFU in a volume of 250 uL (125 uL by nostril) of the recombinant viruses. Twenty-four hours later, naïve guinea pigs (n = 3) were individually placed in the cage of an index animal to assess the potential viral transmissibility by the direct contact (DC) route. Nasal washes were collected from index and contact guinea pigs at 1, 2, 4, 6, 8 and 10 days p.i. and used to determine viral titers and viral loads by TCID50 and qRT-PCR assays, respectively.Six- to eight-week-old female Hartley strain guinea pigs (250–300 g)  were housed in individual cages. Index animals (n = 6) were anesthetized with isoflurane and infected with an intranasal inoculum of 10Serum samples were collected from each guinea pig before inoculation and on day 21 p.i. to evaluate specific antibody levels against influenza B recombinants using standard HAI assays . BrieflyViral PA, PB1 and PB2 genes were amplified by reverse transcription (RT)-PCR from nasal washes collected from animals on day 6 p.i. and sequenced using the ABI 3730 DNA Analyzer .Statistical analyses were performed with GraphPad Prism version 9.1.0. A two-way ANOVA test (with Bonferroni correction) was used to compare viral titers from replication kinetics experiments performed in A549 and Calu3 cells, HAE and nasal washes of guinea pigs.8 and 5.98 × 107 copies/mL, respectively, while WSH19-WT and WSH19-I38T reached a peak at 96 h p.i. with mean titers of 2.26 × 107 and 4.17 × 107 copies/mL, respectively   A. A shar < 0.05) B. By con9 and 5.52 × 108 copies/mL, respectively, while WSH19-WT and WSH19-I38T reached a peak at 120 h p.i. with mean viral loads of 6.29 × 109 and 2.88 × 109 copies/mL, respectively , 96 h p.i.  and 120 h p.i.  while no difference could be seen between the WSH19 recombinant viruses at any time point  (10 TCID50/mL vs. 2.1 log10 TCID50/mL (p < 0.0001) , respectively). Viral titers at other time points were similar between the WT and its respective I38T mutant. At day 8 p.i, 2 of 6 guinea pigs infected with PKT13 I38T were still positive by RT-qPCR vs. only 1 animal in the PKT13 WT group.The impact of the PA-I38T substitution was assessed in experimentally infected guinea pigs. Both infectious viral titers and viral RNA loads were determined in nasal washes from animals infected with WT and PA-I38T recombinant viruses (index) and from their direct contacts (DC) for a period of 10 days post-inoculation (p.i.). Infected guinea pigs (n = 6 per group) shed infectious viruses between days 1 and 6 p.i. for the two genetic lineages A–D. In t < 0.01) A with me6 copies/mL and 4.8 × 106 copies/mL (10 TCID50/mL in both cases on day 2 p.i. (For the B/Victoria lineage (WSH19), viral replication kinetics in guinea pigs were similar between the WT and I38T groups at all time points. The WSH-WT and PA-I38T recombinants reached their peaks at day 2 p.i.  with my 2 p.i. D. At dayA seroconversion was demonstrated for all WT and PA-I38T index animals for the two lineages using HAI assays. HAI titers increased from <10 (on day 0) to 20–320 (on day 21 p.i.). Again, none of the DC animals seroconverted (HAI titers <10) .Until recently, antiviral options against influenza infections were limited to NAIs. Oseltamivir is the most widely used compound from this class owing to its oral bioavailability. Because of structural differences between their NAs, influenza B viruses are less susceptible to NAIs than influenza A viruses, as reflected by higher oseltamivir IC50 values observed for old IBV isolates  and receThe aim of our study was to assess the impact of baloxavir resistance on contemporary influenza B viruses at different levels: in vitro, ex vivo and in vivo. The in vitro and ex vivo experiments were performed by using cells from human origin. This consists of pulmonary  cells and nasal human airway epithelium (MucilAir) to better mimic natural lower and upper respiratory tract conditions. To assess specifically the impact of the I38T substitution, we have chosen to use recombinant viruses as the reverse genetics approach precludes eventual confounding effects of other changes elsewhere in the influenza B genome, such as mutations that may arise in cell-culture passages, in presence of BXA or during BXA treatment of patients. To our knowledge, our study is the first to investigate the infectivity of recombinant PA-I38T influenza B variants using contemporary vaccine strains of B/Victoria and B/Yamagata lineages.In the B/Yamagata background, the PKT13-I38T variant showed reduced viral titers compared to the WT at many time points in A549 and Calu3 cells . HoweverWe further assessed the impact of the I38T PA substitution on virus infectivity in vivo. We are the first group, to our knowledge, to characterize influenza B PA-I38T variants using the guinea pig model. Unlike ferrets, the gold standard animal model to study the influenza virus fitness, guinea pigs are relatively small; hence, easier to handle, inexpensive, and seronegative guinea pigs can be easily obtained, which facilitates statistical analyses . AdditioUnfortunately, we cannot make any conclusion regarding the impact of I38T substitution on viral transmissibility as we found no evidence for viral infection in any DC animals, based on nasal wash viral titers by culture and qRT-PCR. Accordingly, serum samples from DC animals showed HAI titers of <10, similar to pre-infection serum samples . We hypoTaken together, our results combined with those of others suggest that the fitness of BXA-resistant I38T B variants is unlikely to be significantly compromised in most influenza strains. Of note, the relative cost for the I38T PA change was suggested to be higher in A(H1N1)pdm09 than in A(H3N2) viruses .Although the fitness of influenza B variants bearing the PA-I38T substitution seems to be mainly retained in our experiments using pure WT or I38T populations, it would also be interesting to perform competition experiments in vitro and/or in vivo by mixing both WT and PA-I38T viruses in the inoculum and analyzing the evolution of each viral population over time . StudiesIn conclusion, our findings confirm that the relative fitness of BXA-resistant IBVs is relatively conserved but could vary slightly according to the viral strain. It would be of interest to conduct biochemical studies with the influenza endonuclease enzyme. Our study also highlights that surveillance of BXA resistance, which could be induced at high level, is of high importance due to the relatively good fitness of the main I38T variant in the B/Victoria lineage background. Reevaluating dosing regimens and administration schedules of BXA could help reduce the rate of resistance. The combination of BXA with antivirals that have different mechanisms of action, such as a NAIs, could constitute another option to reduce the emergence of resistant variants . The maj


Highly pathogenic avian influenza (HPAI) A/H5N1 viruses continue to pose a significant threat to animal and human health worldwide. In late 2022, the first confirmed case of HPAI A/H5N1 infection in wild birds in Chile near the Chilean-Peruvian border was reported. Active surveillance by our group in the adyacent Lluta river estuary revealed an increase in A/H5N1 prevalence coinciding with the arrival of migratory birds from the Northern Hemisphere. Genomic analysis of A/H5N1-positive samples demonstrated a close genetic relationship to strains detected in Peru during the same period, which originated from A/H5N1 viruses causing outbreaks in North America. Notably, we identified genetic mutations that did not correlate with known enhanced transmission or binding traits to mammalian receptors. In summary, this study provides valuable genomic insights into the A/H5N1 Clade 2.3.4.4b viruses in wild birds in Chile, emphasizing the need for enhanced surveillance and response strategies to mitigate the threat posed by these highly pathogenic avian influenza viruses in South America. Highly pathogenic avian influenza (HPAI) viruses remain a major threat to both animal and human health. Since the emergence of the HPAI A/H5N1 goose/Guangdong (Gs/GD) lineage in 1996, these viruses have spread globally. This includes the recent introduction and reassortment of lineage 2.3.4.4b into the Americas in 2021, impacting both wild and domestic bird populations with high mortality [Pelecanus thagus) at the Lluta River wetland located 10 km south of the Chilean-Peruvian border. We have been conducting active, longitudinal avian influenza (AIV) surveillance in Chile since 2015, with a focus on wild birds and high-risk interfaces with domestic poultry and humans. In anticipation of 2.3.4.4 encroachment into South America, we intensified our ongoing AIV surveillance in the Lluta river estuary  to biweekly sampling in August 2022. AIV prevalence (as measured by M gene specific qRT-PCR) remained below 1% in August and September, 2022. However, prevalence jumped to 2.6% and 11.7% in November and December, respectively, coinciding with the arrival of migratory birds from the Northern Hemisphere. Of the 69 AIV positive environmental fecal samples from November and December , 7 (10.1%) were identified as hemagglutinin (HA) subtype A/H5: 1 obtained in late November, and 6 in December. As per regulations, original samples and RNA were submitted to SAG (submission #230014). Cytochrome Oxidase I speciation [Pelecanus thagus) (n = 1), Franklin’s gull (Larus pipixcan) (n = 1), Grey gull (Leucophaeus modestus) (n = 1), Elegant tern  (n = 2) and Black skimmer (Rynchops niger) (n = 2).On 7th December 2022, the Agricultural and Livestock Service of Chile (SAG) reported the first confirmed case of HPAI A/H5N1 infection in Peruvian pelicans (https://flusurver.bii.a-star.edu.sg/) revealed the presence of several mutation compared to their respective reference strains in the HA, PA and NS gene that could potentially affect host specificity, antigenic escape and/or virulence . Analysis of the internal genes using BLASTn revealed all genes are most similar (>99% nucleotide identity) to A/H5N1 viruses circulating in North and South America .Full genome sequencing of 3 of the samples yielded 2 complete genomes (A/Grey gull/Chile/61947/2022 and A/Black skimmer/Chile/61962/2022) and one partial genome (A/Peruvian pelican/Chile/61740/2022) missing segments 2 and 4 due to low viral load, GenBank accession numbers OQ352540 to OQ352552. The A/H5 HA genes were genetically closest related to Peruvian pelican and chicken samples detected in Peru around the same period, and both Peruvian and Chilean strains derive from A/H5N1 viruses causing widespread outbreaks in poultry and wild birds across North America [Emergence of A/H5 2.3.4.4b in South America poses major risk, as exemplified by the catastrophic impacts to the domestic poultry and wildlife sectors in Europe and North America since 2021 . For theEnhanced surveillance in both domestic and wild birds is critical in Chile and across South America. HPAI A/H5Nx viruses in South America have only been detected in shorebirds and Charadriiformes hosts so far, likely due to passive detection systems. Active, longitudinal surveillance, especially in known AIV hotspots ,8 shouldClick here for additional data file.


Total 25(OH)D, free 25(OH)D, 1,25(OH)2D and the percentage of free 25(OH)D increased after NB-UVB, and PASI and VAS improved. The increase in total and free 25(OH)D remained significant when stratifying for vitamin D confounders. No correlations between disease severity and vitamin D levels were found. Total and free 25(OH)D levels were positively correlated before and after NB-UVB. NB-UVB is an effective treatment for mild to severe plaque psoriasis and increases not only total but also free 25(OH)D levels, as well as the percentage of free 25(OH)D, suggesting an increased bioavailability of skin-produced vitamin D.Vitamin D plays an important role in skin inflammation in psoriasis. The beneficial effects of ultraviolet light B (UVB) phototherapy in psoriasis are partly attributed to UVB-induced increase of vitamin D levels. In clinical practice, total 25-hydroxy vitamin D (25(OH)D) levels are measured to assess sufficiency, but it might be more accurate to measure free 25(OH)D levels. The aim of this study was to measure free serum 25(OH)D levels in psoriasis patients before and after phototherapy and to investigate if free 25(OH)D correlates stronger to disease severity than total 25(OH)D. Twenty adults (>18 years) with psoriasis were included for treatment with narrow-band UVB (NB-UVB) phototherapy for 10–12 weeks. Psoriasis Area and Severity Index (PASI) and Visual Analogue Scale (VAS) were used to assess disease severity. Serum levels of total 25(OH)D, free 25(OH)D, and 1,25(OH) Psoriasis is a chronic, immune-mediated inflammatory skin disease that affects approximately 2–3% of the population worldwide. Patients with psoriasis suffer from substantial morbidity and increased risk of cardiometabolic diseases, mental health disorders, and psoriatic arthritis .It is established that vitamin D plays a role in psoriasis but whether it is involved in its pathogenesis remains unclear ,3. TopicOne possible explanation for the conflicting results could be the use of an inaccurate biomarker for the assessment of vitamin D status in psoriasis patients.According to the free hormone hypothesis, the action of a steroid hormone is mediated through the unbound fraction whereas the protein-bound hormone is biologically inactive. For steroid hormones such as thyroid hormones and sex hormones, the free concentration is measured in clinical practice . In contDBP is the major transport protein for all vitamin D metabolites. The DBP concentration and the protein’s affinity for 25(OH)D define the amount of free 25(OH)D. In conditions such as pregnancy and liver or kidney disease, DBP levels are altered, resulting in a shift of the equilibrium between total and free 25(OH)D .Previously, a mathematical model has been used to calculate the free 25(OH)D level. Since the arrival of a two-step enzyme-linked immunosorbent assay (ELISA) that measures free 25(OH)D directly, studies have shown that the calculated values of free 25(OH)D highly overestimate the measured levels of free 25(OH)D ,8,9.3 is produced from 7-dehydrocholesterol (7-DHC). Although vitamin D can be obtained from the diet, including fortified food items, skin-produced vitamin D constitutes the main source for the body  serum levels, an Electrochemiluminescence immunoassay (ECLIA)  was used. The coefficient of variance (CV) was 12% at 66 nmol/L and 17% at 26 nmol/L. The limit of quantification (LoQ) was <10 nmol/L and >500 nmol/L.To analyze total 25(OH)D [25(OH)DA two-step immunosorbent assay (ELISA)  was used to measure the free 25(OH)D serum levels. The CV was 9% at 4 ng/L. The LoQ was <1.8 ng/L and >40 ng/L.2D. The CV was 10% at 80 pmol/L and 15% at 130 pmol/L. The LoQ was <18 pmol/L.An automated chemiluminescence immunoassay (CLIA) was used to analyze serum 1,25(OH)Serum intact parathyroid hormone (iPTH) was analyzed with ECLIA with an Elecsys PTH STAT . The CV for iPTH was 7% at the level of 3 pmol/L and 3% at 10 pmol/L. The LoQ was <0.5 pmol/L and >530 pmol/L.A detailed description of the analyses, and the method for the calculation of the percentage of free 25(OH)D, are reported earlier .Psoriasis Area and Severity Index (PASI) was used as the investigator-reported outcome measure for disease severity. PASI evaluates the involved body surface area and the appearance of lesions  with a range from 0–72 points, where 0 indicates no disease and 72 maximal diseases. A PASI score ≥ 10 indicates severe disease.The patient-reported outcome measure (PROM) for psoriasis severity was the Visual Analogue Scale (VAS), where 0 corresponds to no psoriasis-related distress, and 10 corresponds to maximal psoriasis-related distress.p < 0.05 was considered statistically significant.Data are presented as mean ± SD, mean (min–max) or median (inter-quartile range (IQR)) if not otherwise stated. Data were analyzed using R version 3.5.3 . Simple descriptive statistics were applied. Spearman’s correlation test was used to test univariate correlations. Wilcoxon’s rank sum test was used for two sample tests. Spearman’s correlation test stratifying with respect to the patient was used when testing for changes over time. Wilcoxon’s signed rank test was used to test differences in PASI and biochemical data before and after NB-UVB. All tests were two-sided and The study was approved by the Ethics Committee at the University of Gothenburg on 22 May 2012 . Declarations of Helsinki protocols were followed. Written informed consent was obtained from all patients.Twenty patients with psoriasis were included. Fifteen patients completed the study. One patient was excluded from the study after the first visit due to a planned sun vacation. The remaining four patients lost to follow-up did not state a reason. Baseline data were analyzed for all twenty included patients.The mean age of the patients was 44.3 ± 15.7 years and the mean disease duration was 19.5 ± 13.5 years.Pretreatment PASI at visit 1 ranged from 3.9–21.0, which corresponds to mild to severe disease. Median PASI was 8.4 (7.6–12.2). Eighteen (90%) patients started NB-UVB phototherapy (visit 1) during winter, as defined in 2 was classified as obesity. Eleven (55%) patients reported arthropathy. Eleven (55%) patients reported psoriasis heredity. Demographic data and possible confounders for vitamin D status are presented in Most subjects had normal blood pressure and were of normal weight. A BMI of >30 kg/m2.The mean number of treatments (patients lost to follow-up excluded) was 26 ± 2.7 and the mean cumulative UVB dose was 36.0 ± 16.2 J/cmp = 0.0001). The mean PASI change was −7.1 ± 4.5. Median VAS decreased from 7.7 (7.0–8.7) to 2.1 (0.9–3.0) (p = 0.002).Median PASI decreased from 8.4 (7.6–12.2) to 2.6 (1.2–3.2) (The effect of NB-UVB treatment on PASI and VAS are presented in 2D levels increased significantly from visit 1 (before therapy) to visit 3 (when treatment was ended). Mean delta 25(OH)D was 50 ± 17 nmol/L and the mean delta free 25(OH)D was 11 ± 4.5 pmol/L. The percentage increase of total 25(OH)D was 95%, free 25(OH)D increased by 124%, 1,25(OH)2D increased by 13%, and the percentage of free 25(OH)D increased by 9.2%, as shown in Total 25(OH)D, free 25(OH)D and 1,25(OH)p = 0.0004), but diastolic blood pressure varied with no clear trend (p = 0.61). iPTH decreased from 3.5 ± 0.7 pmol/L to 2.8 ± 0.6 pmol/L (p = 0.0012) from visit 1 to visit 3.Body mass index (BMI) did not change during NB-UVB treatment. Systolic blood pressure decreased significantly  patients had sufficient levels of 25(OH)D, eight (40%) had insufficient levels, and six (30%) were vitamin D deficient.p < 0.0001) and after NB-UVB (p < 0.003), as shown in Free and total 25(OH)D concentrations were positively correlated at baseline (No correlations were found between disease severity (as determined by PASI and VAS) and levels of any vitamin D metabolite including the percentage of free 25(OH)D at baseline or after NB-UVB. VAS and PASI did not correlate at baseline or after NB-UVB.2D. The percentage increase of total 25(OH)D levels after NB-UVB phototherapy in this cohort was 95%. This is in accordance with previous reports on psoriasis patients after NB-UVB (59–250% increase) [2D, which is the active vitamin D metabolite, was much lower than the increase of 25(OH)D. This is in line with the strict endocrine regulation of the production of the active metabolite to avoid hypercalcemia. Previous studies have reported unaltered levels of 1,25(OH)2D after NB-UVB. However, one study on patients with insufficient levels of 25(OH)D (<75 nmol/L) before phototherapy showed a significant increase of 1,25(OH)2D post-treatment [2. However, this was probably due to the small sample size, To the best of our knowledge, this is the first report on the effects of phototherapy on free 25(OH)D serum levels in psoriasis patients. NB-UVB phototherapy raised serum levels of total 25(OH)D, free 25(OH)D, and 1,25(OH)ncrease) . The increatment ,23. AccoAs expected, NB-UVB was an effective treatment for psoriasis and demonstrated a significant improvement in PROMs and investigator-reported outcome measures. Future studies are needed to evaluate the long-term effects on vitamin D status and disease severity after cessation of phototherapy.Interestingly, NB-UVB also increased the percentage of free 25(OH)D in psoriasis patients. This is a new insight into the biological effects of phototherapy on skin-produced vitamin D, showing that NB-UVB raises the bioavailable vitamin D levels.3, which is the standard treatment for vitamin D insufficiency, has similar effects on the percentage of free 25(OH)D. In a previous study in mice, Duchow et al. reported that skin produced 25(OH)D had a twofold higher bioactivity  compared to oral vitamin D3 [To the best of our knowledge, it is not known whether oral substitution with vitamin Dtamin D3 . An incrPossibly, NB-UVB causes alterations in binding proteins , either by decreased concentrations or altered affinity for 25(OH)D, resulting in a higher proportion of free 25(OH)D. The effects of NB-UVB on serum and tissue levels of DBP in psoriasis patients should be investigated in future studies.The baseline mean percentage of free 25(OH)D was 0.015%, rising to 0.017% after NB-UVB phototherapy. In healthy adults, percentages of free 25(OH)D from 0.02% to 0.09% have been reported [2D) at baseline or after NB-UVB). Thus, the hypothesis that free 25(OH)D correlates better with disease severity than total 25(OH)D could not be confirmed in this small cohort. However, it is important to note that the correlation between changes in 25(OH)D levels and psoriasis improvement is still not clear, but the evidence implies that NB-UVB therapy can provide multiple benefits for psoriasis patients.We found no correlation between psoriasis severity and any of the vitamin D metabolites which were measured D and 1,25(OH)Free and total serum 25(OH)D levels were positively correlated at baseline and after NB-UVB. This suggests that the accepted marker for vitamin D status, total serum 25(OH)D, is relevant for assessing vitamin D status in individuals with psoriasis. However, there was an increased dispersion of corresponding values for free and total 25(OH)D after NB-UVB compared to baseline. This could indicate an alteration in the equilibrium between free and total 25(OH)D as a result of NB-UVB phototherapy.In line with previous results by Osmancevic et al., serum iPTH decreased significantly after phototherapy . It has An inverse correlation between the percentage of free 25(OH)D and total 25(OH)D has been reported before and suggests a compensating mechanism altering the relationship between free and total 25(OH)D when needed ,26.Decreased blood pressure as a result of UVB exposure was expected and has also been shown before . HyperteThe main limitation of this study is the small number and the heterogenicity (regarding disease severity) of participants. Twenty patients were included of which 15 completed the entire protocol. Hence, the results must be interpreted with caution. The method used for measuring 25(OH)D levels (ECLIA) is not the gold standard method, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), but it is still a validated method and widely used in the assessment of vitamin D status . To eval2D in psoriasis patients and is a clinically effective treatment. The effect on vitamin D metabolites is particularly relevant for psoriasis, as vitamin D might play a role in the pathogenesis. The increase of 25(OH)D levels after NB-UVB in this cohort was comparable to previous reports. The increase of total and free 25(OH)D remained significant in sub-analyses when stratifying for confounding factors for vitamin D status. We found no correlation between disease severity and vitamin D levels in this small cohort. The percentage of free 25(OH)D increased after NB-UVB implying that 25(OH)D not only increases but also becomes more bioavailable. This might be unique for UVB-induced vitamin D skin synthesis suggesting that NB-UVB therapy can provide multiple benefits for psoriasis patients. Total 25(OH)D seems to be a relevant marker for vitamin D status in psoriasis patients, but larger studies are needed to test the free hormone hypothesis in this group.NB-UVB phototherapy increases serum levels of total 25(OH)D, free 25(OH)D, and 1,25(OH)


Sphingosine-1-phosphate (S1P) is an important sphingolipid molecule involved in regulating cardiovascular functions in physiological and pathological conditions by binding and activating the three G protein-coupled receptors  expressed in endothelial and smooth muscle cells, as well as cardiomyocytes and fibroblasts. It exerts its actions through various downstream signaling pathways mediating cell proliferation, migration, differentiation, and apoptosis. S1P is essential for the development of the cardiovascular system, and abnormal S1P content in the circulation is involved in the pathogenesis of cardiovascular disorders. This article reviews the effects of S1P on cardiovascular function and signaling mechanisms in different cell types in the heart and blood vessels under diseased conditions. Finally, we look forward to more clinical findings with approved S1PR modulators and the development of S1P-based therapies for cardiovascular diseases. Sphingosine-1-phosphate (S1P) was discovered in the 1960s as a metabolic product of sphingolipids . Then, sCardiovascular disease (CVD) is one of the leading causes of death in the world. The pathogenesis and treatment of CVD have always been a difficult and hot topics in cardiovascular research. Recently, abnormal levels of sphingolipid molecules have been detected in the plasma or blood vessels of patients with atherosclerosis, hypertension, and other CVD ,8, indic−/− mouse plasma has undetectable Sphk activity and that S1P levels were reduced by approximate 65%. They speculated that the Sphk1a isoform might contribute to the establishment of an S1P gradient in the vasculature. Nagahashi et al. [Homeostatic S1P levels are maintained through their generation, transport, and degradation . SphKs mi et al.  confirmei et al. .S1P is mainly derived from erythrocytes, endothelial cells (ECs), and platelets and maintains high concentrations in the blood and lower levels in other tissues . S1P levi/o, Gαq, Gα12/13, and Gαs). Gαi/o extensively couples with S1PR1-5 and facilitates the activation of the small GTPases Ras and Rac, phosphatidylinositol-3-kinase (PI3K)/Akt, and phospholipase C (PLC) to promote cell proliferation, migration, apoptosis inhibition, and intracellular free calcium as well as inhibit adenylyl cyclase activity to reduce cAMP [12/13 and Gαq couple with S1PR2/3/4/5 and S1PR2/3, respectively, which primarily activate the small GTPase Rho/ROCK and PLC pathways to inhibit migration [s act as relay mediators of S1PR1 signaling to control vascular integrity [+ current or the AKT and ERK pathways in cardiomyocytes [As S1P acts predominantly through the activation of different subtypes of S1PRs, the spatial distribution of S1PRs is a major determinant of S1P’s versatile roles in physiological processes. It has been reported that S1PR1, 2, and 3 are ubiquitously expressed in all tissues and organs, including the cardiovascular system . There auce cAMP . Gα12/13igration . Other sntegrity . In addimyocytes ,31. Howei complex [13 complex [i/inverse agonist [The cryo-electron microscopy (cryo-EM) structures of the S1PR1-Gα complex ,33,34, S complex , S1PR3-S complex , and S1P agonist ,38 have  agonist . These fThe effects of S1P on the cardiovascular system occur mainly through S1PR1-3 and downstream signaling pathways . S1P trai to activate the small GTPase Rac/Cdc42 or the PI3K/ERK pathway [In ECs, S1P plays an important role in endothelial barrier maintenance, vasculogenesis, vascular tone, inflammation, lymphocyte trafficking, chemotaxis, and immunity via its receptors S1PR1-3 ,40. The  pathway . Liu et  pathway  reported pathway . i signaling during tumor growth [The endothelial glycocalyx (EG) is a jelly like protective layer covering the luminal surface of the endothelium and forms part of the barrier . Plenty r growth . Moreover growth . Wang etr growth  found thr growth . Studiesr growth . 2O2-mediated APE/Ref1 translocation, induces VSMCs migration and vascular neointima formation [In vascular smooth muscle cells (VSMCs), it has been reported that elevated S1PR1 expression in response to angiotensin II (Ang II), via Hormation . Kerage ormation  used eNOormation . Howeverormation . Circulating S1P could also produce natriuretic effects by acting on epithelial sodium channels via activation of S1PR1 in the renal medulla, causing significant increase in urine flow and sodium excretion , and it i to reduce cAMP accumulation-associated decrease in L-type calcium channel current, or coupling Gβγ to activate inwardly rectifying K+ current and shortening action potential duration (APD) [i pathway activates AKT and ERK in adult mouse ventricular myocytes to inhibit isoproterenol-stimulated cAMP accumulation, leading to a negative inotropic response [It has been established that S1P affects cardiac muscle contraction, heart rate, and cardiac fibroblasts. S1PR1-3 are the main isoforms of S1PRs expressed in the heart, and the expression patterns of these receptor subtypes in different cardiac cell types directly reflect differential regulatory effects and cardiac performance . It is ron (APD) . Anotherresponse . Cardiomresponse .If) and the voltage-gated calcium current (IL-Ca) in rabbit sinoatrial node cells and mediates the decrease in heart rate only in the presence of the beta-adrenergic agonist isoproterenol [S1P also inhibits heart rate, mainly by activating S1PR1. Several studies have reported that long-term treatment with S1PR modulators could reduce cardiac autonomic activation and decrease heart rate variability by binding S1PRs to trigger G protein-gated inwardly rectifying potassium (GIRK) channels on cardiac myocytes ,69. Atrioterenol . TherefoAtherosclerosis (AS) is characterized by the focal accumulation of lipids in arterial walls and is identified as the effect of hemodynamic wall shear stress (WSS) on endothelial permeability. In recent years, it has been found that S1P is associated with HDL particles and produces antiatherosclerotic effects ,72,73. SAngiogenesis refers to the process of endothelial cell proliferation and migration, which depends on pro- and anti-angiogenic molecules. Well-regulated angiogenesis plays a pivotal role in many physiological conditions such as embryonic development, wound healing, and endometrial changes in the menstrual cycle. Angiogenesis is also the basis of various pathological processes, including tumor metastasis and atherosclerotic plaque formation . It has Ca2.3 and KCa3.1 channels in ECs. Blocking KCa2.3 and KCa3.1 channels attenuated the vasodilation response caused by acute S1P stimulation of the mesenteric arteries [Several studies have revealed that S1P/S1PRs signaling promote both vasorelaxation via ECs and vasoconstriction by VSMCs, thus regulating blood pressure in a balanced way. This process involves not only store-operated calcium channels (SOCs) in VSMCs  but alsoarteries . Aldostearteries . Treatmearteries . The admarteries . Interesarteries . The rolS1P is an important endogenous cardioprotective agent in myocardial ischemia and reinfusion (I/R) injury . S1P mayMyocardial infarction (MI) is caused by acute coronary occlusion and leads to myocardial injury in the corresponding area of the blood supply . After M12/13 [2 (PGE2) generation, which eventually results in apoptosis of human cardiac fibroblasts [Fibroblasts are an important cell type in the heart and appear to be an important source of endogenous S1P in the heart with higher levels of SphK activity than cardiomyocytes . Studies12/13 . The Rho12/13 . Rho kin12/13 . Fibrobl12/13 . It is a12/13 . Moreove12/13 . A recenroblasts . During myocardial remodeling, the S1P/S1PR1 pathway promotes reparative macrophage proliferation  and tighS1P is a biologically active lipid molecule involved in the regulation of various cardiovascular functions. The differential expression of its three receptors (S1PR1-3) in different cell types, along with the diversity of G proteins they couple, determine the downstream effects of S1P. S1P synthase, transporters, and metabolic enzymes also affect cardiovascular function by the dynamic control of S1P levels. Due to the complex regulatory network, S1P signaling shows multifaceted and sometimes opposite effects on the cardiovascular system. Overall, S1P is a cardioprotective factor against several types of myocardial injury and can play either a beneficial or harmful role in vascular diseases . In rece