The binding of p-sulfonylphenoxy moieties to the phthalocyanine structure favors excellent solubilities in water, as well as providing an inhibition effect on carbonic anhydrase (CA) I and II isoenzymes and paraoxonase (PON1) enzyme. According to biological activity results, both complexes inhibited hCA I, hCA II, and PON1. Whereas1and2showed moderate hCA I and hCA II (off-target cytosolic isoforms) inhibitory activity , they exhibited strong PON1 (associated with high-density lipoprotein [HDL]) inhibitory activity . The inhibition kinetics were analyzed by Lineweaver–Burk double reciprocal plots. It revealed that1and2were noncompetitive inhibitors against PON1, hCA I, and hCA II. These complexes can be more advantageous than other synthetic CA and PON inhibitors due to their water solubility. Docking studies were carried out to examine the interactions between hCA I, hCA II, and PON1 inhibitors and metal complexes at a molecular level and to predict binding energies.The investigation of carbonic anhydrase and paraoxonase enzyme inhibition properties of water-soluble zinc and gallium phthalocyanine complexes ( Phthalocyanines (Pcs), an important family of porphyrinoid complexes, have many applications such as gas sensors [1], solar cells [2], liquid crystals phases , electrochromic materials [5], and photosensitizer [6–9] in photodynamic therapy (PDT). For these applications, the photophysical and photochemical features of phthalocyanines can be fine-tuned by the introduction of various substituent groups. It is also known that the physical and chemical properties of the complex depend significantly on the nature of the metal atom coordinated to the phthalocyanine ring . The lack of water solubility of phthalocyanines limits their use in many areas. Also, water solubility is very important for cancer treatments because the complexes are injected into the patient’s bloodstream with a hydrophilic system [12]. Water-soluble phthalocyanines continue to attract attention to their interactions with DNA and their ability to trigger DNA photodamage by accumulating in many cancer cells. Sulfonate, carboxylate, and phosphorus groups can be used from anionic substituents to the macrocyclic ring by various intermediates or directly attached to the phthalocyanine complexes. In particular, the binding of sulfonic acid groups to the phthalocyanine ring has two important effects: they increase the effectiveness of their antitumor properties [13], and by inducing the repulsion of phthalocyanine rings, they become water-soluble as monomers . Also, in the literature, gallium and zinc metal complexes of the phthalocyanines are examined due to their superior antitumor properties [13]. Thus, these phthalocyanines are beneficial to PDT and various biological applications.Carbonic anhydrase (CA) isoenzymes are metalloenzymes that catalyze a very easy reaction: the hydration of CO2 to bicarbonate and H+ [15–17]. This key reaction plays a significant role in more pathological and physiological mechanisms associated with ion transport, pH control, and fluid secretion [18]. The inhibition of these isoenzymes is the main goal correlated with the treatment of diverse diseases such as obesity, glaucoma, and epilepsy. More recently, CA inhibition was validated as a novel approach to fighting metastases and tumors. Paraoxonase, which has an important role in living metabolism, is a calcium-dependent enzyme and is also an organophosphate hydrolyzer. These enzymes can hydrolyze aromatic carboxyl esters such as phenylacetate and various lactones. The name “PON” is derived from paraoxon, a common in vitro substrate. Paraoxonase 1 (PON1), which is associated with high-density lipoprotein (HDL) with 355 amino acids, is the most studied member of the mammalian enzyme family. PON1 is considered an important enzyme for two main reasons in metabolism: (i) it protects the system against the neurotoxicity of organophosphates and (ii) oxidizing lipid levels interfere with the onset of atherosclerosis, thus preventing oxidation of low-density lipoproteins, so the activity of PON 1 is considered a risk for atherosclerosis.Taking these properties into consideration, we focused on water-soluble complexes as a strategy to explain Pcs in conjunction with the sulfonic acid group to study enzyme inhibition, chemical interactions, and theoretical properties. In this regard, water-soluble sulfonated zinc and gallium phthalocyanine complexes (1 and 2) were synthesized and their inhibition potential of hCA I and II isoenzymes and paraoxonase enzyme were investigated to give direction for further studies. Also, metal effects on carbonic anhydrase and paraoxonase inhibition activity were examined. To the best of our knowledge, even though there are some studies in the literature in which phthalocyanines are evaluated as carbonic anhydrase and α‐glucosidase enzyme inhibitors [19–22], carbonic anhydrase and paraoxonase inhibition activity of anionic water-soluble phthalocyanines has not been reported. Consequently, this is the first study including the evaluation of anionic water-soluble phthalocyanines bearing Ga and Zn metals and the sulfonic acid group as both potential carbonic anhydrase and paraoxonase enzyme inhibitors. Furthermore, molecular docking studies were also applied for a better understanding of the structural and binding profiles of synthesized complexes at the active sites of target enzymes.Details on equipment, materials, enzyme inhibition, and molecular docking parameters are supplied as Supporting Information. Water-soluble zinc and gallium phthalocyanine complexes (1 and 2) were prepared according to the reported procedure . Biological activity assays and IC50 graphs are provided in Supporting Information.Figure 1 shows the molecular structure of nonperipherally substituted sulfonated zinc and gallium phthalocyanine complexes (1 and 2).The solution spectra show a spectral characteristic indicating D4h symmetry, which is typical for Pc complexes [24]. The absorption peak in the near-UV region is the B-band or Soret band that is attributed to the a2u→e*g transition; a further band in the visible region is related to the Q-band caused by the p–p* transition a1u→e*g. It can be stated that there are some vibrational bands at relatively shorter wavelengths that are a standard property of metallophthalocyanines [25]. The UV-vis absorption spectra of nonperipherally substituted phthalocyanine complexes in DMSO shows two main peaks, the characteristic ligand centered π–π* transitions of a monomeric zinc and gallium phthalocyanine derivatives (1 and 2) with Q-band maxima at 695 and 711 nm, respectively  .The CA I and CA II inhibitory activities of the synthesized complexes were determined by hydratase activity (used carbon dioxide as a substrate) and esterase activity (used 4-nitrophenyl-acetate [NPA] as a substrate) assays to calculate the inhibition constants (Ki). Ki values were calculated from the Lineweaver–Burk graphs . The Ki values of the synthesized compounds 1–2 against PON1, hCA I, and hCA II isoforms are given in Table 1. Complexes 1 and 2 inhibited the cytosolic isoforms hCA I and hCA II in the micromolar range . Complex 1, which included Zn metal, had higher inhibitory activity against both hCA I and hCA II with the Ki of 26.09 µM and 30.95 µM, respectively than complex 2 containing Ga metal . According to these results, it could be considered that the atomic diameter of the Zn metal is larger than Ga; thus, the Zn complex is bulkier than the Ga complex. Their different steric effects could change the inhibitory activity because of an entrance of the active site cavity and the van der Waals interactions with amino acid residues.Acetazolamide (AAZ) is one of the best-known CAIs and is also used as a standard in CA assays [27]. Whereas complexes 1 and 2 showed much weaker inhibitory activity against hCA I and hCA II than AAZ  , they exhibited higher hCA I and II inhibitory activity than some synthesized compounds  reported as CAIs in the literature [30–33].The sulphonamides (known as strong CAIs) bind in the deprotonated form to the catalytically critical Zn (II) ion in the enzyme active site , also contributing an extensive hydrogen bond and van der Waals interactions with amino acid residues of the enzyme active site, as reported in X-ray crystallographic studies of enzyme-inhibitor complexes [36]. We consider that the –SO3H moieties of the synthesized complexes can interact with Zn (II) ion and form the hydrogen bonds with amino acid residues in the enzyme active sites.On the other hand, the in vitro inhibition effects of synthesized complexes on paraoxonase 1 (PON1) were investigated using paraoxon as a substrate. Both complex 1 and 2 inhibited PON1 (associated with HDL) with Ki of 0.37 µM and 0.27 µM, respectively, as noncompetitive inhibitors . These results demonstrated that complex 1 and 2 have a much stronger PON1 inhibitory activity than the reported PON1 inhibitors  in the literature [37–39]. Besides, the changing metals and their atomic diameters have not shown a significant effect on PON1 inhibition.Docking calculations were carried out using the HEX 8.0.0 program [40]. This docking simulation program has allowed the calculation of drug candidate molecules for high atomic weight with a metal center. Docking calculation parameters are correlation type, compute device, FFT mode, and sampling method. These parameters are shape only, CPU, 3D, and range angles, respectively. Grid parameters are solution-100 and step size-. Docking simulations for investigated complexes were implemented against 4WR7, 5AML, and 1V04 target proteins representing hCA I, hCA II, and PON1 enzymes, respectively. 4WR7 is the crystal structure of human carbonic anhydrase isozyme I with 2,3,5,6-tetrafluoro-4-(propylthio) benzenesulfonamide. CA is an enzyme that catalyzes reversible carbon dioxide hydration. It is a metalloenzyme that ensures the regulation of acid-base balance and ion transport in all tissues and organs. It is known that increased levels of different CAs are associated with various diseases such as epilepsy and cancer. Since the enthalpy and entropy distribution range of this protein is quite wide, it has a greater entropy contribution to the binding affinity during simulation than enthalpy [41]. 5AML is the three-dimensional structure of human carbonic anhydrase II in complex with 2-(but-2-yn-1-ylsulfamoyl)-4-sulfamoylbenzoic acid. The 5AML target protein has selectivity corresponding to some CA isoforms with medical applications. This protein has a high potential modulus as it contains aliphatic, alkenyl, aralkyl groups, and saccharin derivative substituents [42].PON1 is a glycoprotein Ca2+-dependent ester hydrolase, which is synthesized in the liver and is found in HDL in human serum, consisting of 355 amino acids [43]. PON1 is an enzyme with paraoxonase, arylesterase, and lactonase activities that can react with a wide variety of substrates [44]. The three-dimensional structure of a hybrid mammalian recombinant PON1 variant obtained by directed evolution (rePON1) was recently determined providing the first structural information about this hydrolase family, and this structure was named 1V04 [45]. The biological activity of synthesized zinc and gallium phthalocyanine complexes (1) and (2) against hCA I, hCA II, and PON enzymes was attempted to be elucidated by molecular simulation method. Secondary chemical interactions between the amino acid residues of the 4WR7, 5AML, and 1V04 target proteins and the complexes studied were also investigated. In the coupling studies, the estimated free energy of the binding values and the binding modes of the target proteins and metal complexes is given in Table 2 and Figure 5, respectively. According to the docking results in Table 2, the inhibition activities between the complexes and the target proteins, the gallium complex (2) is greater than the inhibition activity of the zinc complex (1). Gallium complex is located in the symmetry cavity of the 4WR7 target protein. Since the gallium complex contains chlorine atoms, it contains a halogen bond in the secondary chemical interaction type. The halogen bond occurs between the chlorine atom bound to the gallium metal and the Gln92 amino acid residue. Also, the gallium complex is in polar interaction with the target protein.The increase in the number of secondary chemical interactions between this complex and target proteins may have increased the estimated binding energy. It is clear from the results that in the gallium phthalocyanine complex there are two H-bonds between the His94 and Thr199 amino acid residues of the 4WR7 target protein. This is seen as an H-bond in the zinc complex. The H-bond appears between the zinc phthalocyanine complex and the His199 amino acid residue of the 4WR7 target protein. Both metallophthalocyanine complexes show a similar trend with the 5AML target protein and the 4WR7 target protein. However, in contrast to the interaction of complexes with 5AML target protein, polar interaction is unlike any other.Zinc and gallium complexes are in a stronger interaction with the 1V04 target protein, which represents the PON1 enzyme, compared to other enzymes. The types of secondary chemical interactions of the compounds with the 1V04 target protein are greater, as seen in Table 2. In the gallium metal-centered complex, unlike in the zinc complex, the halogen bond with the HIS115, LEU240, LEU267, and ILE291 amino acid residues draws attention. The binding energies obtained from the docking results show that the calculated energy values are in a trend parallel with enzyme activities.As a result, simulation results tend to be similar to experimental inhibition activity. Docking studies are thought to be very important for understanding the chemical interaction mechanism in the inhibition effect.In this paper, the investigation of carbonic anhydrase and paraoxonase enzyme inhibition properties of water-soluble sulfonated zinc and gallium phthalocyanines are reported for the first time. The results showed that complex 1 and 2 inhibited the cytosolic isoforms hCA I and hCA II (off-target cytosolic isoforms) in the micromolar range . Moreover, they inhibited PON1 (associated with HDL) with Ki of 0.37 µM and 0.27 µM, respectively. The inhibition kinetics was analyzed by Lineweaver–Burk double reciprocal plots. The analysis revealed that complex 1 and 2 were noncompetitive inhibitors against PON1, hCA I, and hCA II. Whereas complex 1 and 2 showed moderate hCA I and hCA II inhibitory activity, they exhibited strong PON1 inhibitory activity. Furthermore, the changing metals (Zn and Ga) and their atomic diameters affected the CA inhibitory activity, while they did not show a significant effect on PON1. These complexes can be more preferable than other synthetic CA and PON inhibitors due to their high water solubility. Finally, the inhibition efficacy between zinc and gallium complexes and hCA I and hCA II enzymes has been studied in detail with molecular simulation, and experimental data and docking results are highly compatible.


This work utilizes X-ray crystallography to provide mechanistic insights into how carbonic anhydrase II can act as a nitrite reductase in the presence of copper. 2−) is utilized as a source of NO through enzymes known as nitrite reductases. These enzymes reduce NO2− to NO through a catalytic metal ion, often copper. Recently, several studies have shown nitrite reductase activity of mammalian carbonic anhydrase II (CAII), yet the molecular basis for this activity is unknown. Here we report the crystal structure of copper-bound human CAII (Cu–CAII) in complex with NO2− at 1.2 Å resolution. The structure exhibits Type 1 (T-1) and 2 (T-2) copper centers, analogous to bacterial nitrite reductases, both required for catalysis. The copper-substituted CAII active site is penta-coordinated with a ‘side-on’ bound NO2−, resembling a T-2 center. At the N terminus, several residues that are normally disordered form a porphyrin ring-like configuration surrounding a second copper, acting as a T-1 center. A structural comparison with both apo-  and zinc-bound CAII (Zn–CAII) provides a mechanistic picture of how, in the presence of copper, CAII, with minimal conformational changes, can function as a nitrite reductase.Nitric oxide (NO) promotes vasodilation through the activation of guanylate cyclase, resulting in the relaxation of the smooth muscle vasculature and a subsequent decrease in blood pressure. Therefore, its regulation is of interest for the treatment and prevention of heart disease. An example is pulmonary hypertension which is treated by targeting this NO/vasodilation pathway. In bacteria, plants and fungi, nitrite (NO The active site is divided into a distinct hydrophobic and hydrophilic side. The hydrophobic side  stabilizes the COet al., 2008et al., 2008et al., 2012In humans, the most common source of NO is its synthesis by endothelial nitrogen oxide synthase (eNOS), which catalyzes the oxidation of arginine to produce NO and citrulline  , which can replace the zinc in the CAII active site, as previous research has shown that CAII preferentially binds copper with 50-fold specificity over zinc  site and coordinated by a cysteine, a methionine and two histidines, functions to transfer electrons to the second copper site termed the Type II (T-2) site , and a nitrite molecule, bound in a ‘side-on’ conformation, coordinated via an oxygen and a nitrogen Fig. 1. The copa)]. In the active site, the Cu–CAII has a slightly altered water network compared with the Zn–CAII structure [Fig. 1b)] in that the zinc-bound solvent is replaced with a bound NO2− molecule in a ‘side-on’ configuration . However, previous structural studies of bacterial copper nitrite reductases have revealed endogenous ligands bound to the T-2 site . While the origin of these ligands is unknown, others have hypothesized synchrotron radiation as a possible source of high-energy ions leading to the formation of these molecules ]. The CO2 binding shares the same hydrogen bond with Thr199 while also forming hydrophobic interactions with Val121, Val143 and Trp209 [Fig. 2a)]. The Cu–CAII active site retains the same W1, W2, W3a and W3b positions as Zn–CAII ]. Presumably, the additional water molecules complete a solvent network to span the two copper-binding sites, allowing the electron transfer necessary for the nitrite reductase reaction [Fig. 1b)].As mentioned previously, an ordered water network exists within Zn–CAII, responsible for rapid proton transfer  . However, in the Cu–CAII structure, the N terminus becomes ordered, forming a pseudo-porphyrin ring, with the copper coordinated by several nitrogens [Fig. 3a)]. Previous work using paramagnetic nuclear magnetic resonance (NMR) techniques and X-ray absorption spectroscopy predicted this N-terminal structure as an amino terminal copper and nickel (ATCUN) binding motif as a high-affinity binding site for copper, Kd ≃ 0.5 nM ]. It is more than likely that this site serves as the site of electron transfer to the T-2 copper active site [Fig. 1b)]. The pseudo-porphyrin ring conformation, formed by the Cu–CAII N terminus has a striking resemblance to that of heme-containing nitrite reductases [Fig. 3b)]. Structural superposition of the Cu–CAII N terminus with Pseudomonas aeruginosa nitrite reductase heme gave a root-mean-square deviation (RMSD) of 0.3 Å [Fig. 3c)]. While not a porphyrin ring structure, this pseudo T-1 site, which has not previously been observed in metal–CAII structures, would provide the necessary electron donor site required for nitrite reduction. Hence, this structure provides an obvious mechanism for Cu–CAII to function as a nitrite reductase. Based on published mechanistic studies with bacterial nitrite reductase, the Cu–CAII active site has the T-1 and T-2 sites, the necessary bridging waters, and acidic residues to stabilize NO2− and its intermediates, thus catalyzing nitrite reduction  not observed in the other CA isoforms  of Cu–CAII.While it is accepted that zinc-bound CAII exists in the blood, there is currently no direct experimental evidence to suggest the existence of copper-bound CAII. However, previous work from Aamand et al. and the affinities and concentrations from above, we can predict that ∼86% of CAII in the blood will have copper bound in the canonical active site  and 1 mM zinc sulfate and incubated for an additional 3 h. The zinc was added to aid in protein expression and folding, thus improving the yield. The cells were pelleted via centrifugation and subsequently lysed using a microfluidizer set to 18 000 PSI (1 PSI ≃ 6895 Pa). The Zn–CAII was purified from the cell lysate using affinity chromatography with a p-aminomethyl-benzenesulfonamide affinity column. The final protein stock was buffer exchanged with storage buffer  using a centrifugal filter. Purity was determined with SDS–PAGE and protein concentration was determined by UV–Vis spectroscopy at 280 nm.Human CAII was expressed and purified according to previously published protocols . The solution was then gently stirred overnight at room temperature (20°C) and then passed over the p-aminomethyl-benzenesulfonamide affinity column. Any residual Zn–CAII attached to the column, while the apo-CAII was collected in the flow through. The apo-CAII was buffer exchanged using centrifugal filters against storage buffer to remove any residual chelating agent. The loss of enzyme activity was verified using a standard colorimetric esterase based kinetic method, as described elsewhere .The crystals were harvested utilizing MiTeGen loops, flash cooled in liquid nitrogen and shipped to Stanford Synchrotron Radiation Lightsource (SSRL). Data were collected at the 9-2 beamline at SSRL using a PILATUS 6M detector with 0.3° oscillations, a wavelength of 0.9795 Å and a detector distance depending on the resolution of the crystal diffraction. Each dataset consisted of 600 images for a total of 180° of data. X-ray absorption spectroscopy was also performed at the 9-2 beamline to determine the presence of copper or zinc in the respective crystals (XDS then merged and scaled to the P21 space group using the program AIMLESS via the CCP4 program suite (Kabsch, 2010Phenix (Liebschner et al., 20193ks3 (Avvaru et al., 2010et al., 2010Phenix, while the program Coot was utilized to add solvent molecules and make individual real-space refinements of each residue when needed (Adams et al., 2010PyMOL and protein–ligand interactions and bond lengths were determined using LigPlot+ (Schrödinger, LLC; Laskowski & Swindells, 2011Table S1 in the supporting information. The apo- and Cu–CAII structures have been deposited in the PDB with accession numbers 6pea and 6pdv, respectively.The diffraction images were indexed and integrated using 10.1107/S2052252520000986/lz5032sup1.pdfSupporting table and figures. DOI: copper–carbonic anhydrase II, a nitrite reductase, 6pdvPDB reference: high-resolution apo-carbonic anhydrase II, 6peaPDB reference:


II compounds are widely used in catalysis, photocatalysis, and medical applications. They are usually obtained in a reductive environment as molecular O2 can oxidize RuII to RuIII and RuIV. Here we report the design, identification and evolution of an air‐stable surface [bipy‐RuII(CO)2Cl2] site that is covalently mounted onto a polyphenylene framework. Such a RuII site was obtained by reduction of [bipy‐RuIIICl4]− with simultaneous ligand exchange from Cl− to CO. This structural evolution was witnessed by a combination of in situ X‐ray and infrared spectroscopy studies. The [bipy‐RuII(CO)2Cl2] site enables oxidation of CO with a turnover frequency of 0.73×10−2 s−1 at 462 K, while the RuIII site is completely inert. This work contributes to the study of structure–activity relationship by demonstrating a practical control over both geometric and electronic structures of single‐site catalysts at molecular level.Ru II(CO)2Cl2] single site is designed towards CO oxidation, while all other RuIII single sites are not active. The methodology is further extended to nine transition‐metal single‐site systems, enabling the use of surface coordination chemistry in heterogeneous catalysis.An air‐stable surface [bipy‐Ru II complexes were first discovered in the 19th century.II compounds function as reducing agents and only a few RuII complexes with strong‐field π‐acceptor ligands are air‐stable.[E⊖ of RuIII/RuII in [Ru(H2O)6]3+/ [Ru(H2O)6]2+, [Ru(NH3)6]3+/ [Ru(NH3)6]2+, [Ru(en)6]3+/ [Ru(en)6]2+, [Ru(CN)6]3−/ [Ru(CN)6]4− and [Ru(bipy)6]3+/ [Ru(bipy)6]2+ pairs are 0.23 V, 0.10 V, 0.21 V, 0.86 V and 1.24 V, respectively.II system can be oxidized by molecular O2 at elevated temperatures, limiting its study and application in heterogeneous oxidation chemistry.Rur‐stable.1, 5 T6]3+/[Ru(bipy)6]2+ pair, we covalently bind bidentate bipyridine (bipy) ligand into the polyphenylene (PPhen) framework via a C−C coupling reaction, converting the bipy ligands from solute molecules into surface binding sites. This enables the design of surface single‐site and single‐atom catalysts, which mimic molecular catalysts and have unique coordination and electron configuration compared with particles and clusters.3OH steam reforming,II(CO)2Cl2] single site via simultaneous RuIII to RuII transition and Cl−/CO ligand exchange. Such solid/gas interface coordination chemistry, including both geometric and electronic structures, is monitored by a combination of in situ X‐ray absorption fine structure (XAFS), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), ex situ far‐FTIR, X‐ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations. The [bipy‐RuII(CO)2Cl2] site is stable in air for at least 400 days. It enables CO oxidation with a turnover frequency (TOF) of 0.73×10−2 s−1 at 462 K, while the RuIII site is completely inert. Such distinct catalytic performance could result from the geometric configuration of the [bipy‐RuII(CO)2Cl2] site and its optimized energy level that boost CO conversion.Inspired by the relatively high reduction potential of [Ru(bipy)4]−, [bipy‐CoCl2], [bipy‐Ni(H2O)4]2+, [bipy‐CuCl2], [bipy‐Cu(H2O)4]2+, [bipy‐ZnCl2], [bipy‐RuCl4]−, [bipy‐RuBr4]−, [bipy‐RhCl4]−, [bipy‐PdCl2], and [bipy‐PtCl4]. This variety of selection including both metal cations and ligands suggests great potential in catalysis and mechanistic understandings.Building a surface single site on PPhen with bipy ligands is proven to be a general and versatile method. Eleven types of uniform and well‐defined surface single sites are obtained, including − single site in the presence of HCl, in which the bipy ligand anchors the Ru3+ while Cl− anions are balancing the +3 charge. High density and good dispersion of single sites are achieved, with more than 200 Ru ions identified over 40 nm2 of high angle annular dark field‐scanning transmission electron microscopy (HAADF‐STEM) image .IIICl4]− is stable in air up to 350 °C , suggesting its capability for reactions at elevated temperature even in oxidative environments. The 5.8 wt % Ru loading is calculated from the difference in weight residue between the PPhen‐[bipy‐RuIIICl4]− and PPhen‐bipy. The value is smaller than the circa 8.6 wt % Ru loading calculated from the ratio of the bipy ligand in the PPhen‐bipy . The possible reasons are the loss of bipy during PPhen‐bipy synthesis and the formation of inaccessible bipy site. The BET equivalent surface area is 465 m2 g−1, providing the porous framework for heterogeneous catalysis . Obtaining such well‐defined [bipy‐RuIIICl4]− site is crucial for determining the solid/gas interface coordination chemistry, which provides the possibility to trace further structure evolution.The PPhen‐bipy reacts with RuCle Figure . The loaI Figure . The extI Figure . The coo2 reduces [bipy‐RuIIICl4]− into Ru nanoparticles , owing to the fact that H2 takes away Cl‐ and no other ligand is available at the surface to maintain the hexacoordinated octahedral structure. Consequently, CO, as a reductive gas phase ligand, is selected for this ligand exchange reaction at the solid/gas interface. O2 is co‐fed, thereby any detection of the RuII will guarantee its stability under molecular O2. At room temperature, the initial [bipy‐RuIIICl4]− remains unchanged , showing no ligand exchange with CO. The release of HCl is observed starting from 433 K ‐[Ru(bipy)(CO)Cl3] .mer(Cl)‐[Ru(bipy)(CO)Cl3] .III . We conclude that majority of the RuIII surface complex becomes mer(Cl)‐[bipy‐RuIII(CO)Cl3] via:HK Figure . A surfa1 Figure . This vimer(Cl)‐[bipy‐RuIII(CO)Cl3] is relatively stable because any removal of the Cl− will result in the formation of unfavorable positively charged Ru complexes. With 100 mg of PPhen‐[bipy‐RuIIICl4]H, 1.25 mL of CO is consumed, corresponding to 5.6 wt % Ru loading on the initial PPhen‐[bipy‐RuIIICl4]H . This observation is in good agreement with the 5.8 wt % Ru loading calculated from the Thermogravimetric analysis (TGA), proving the chemical stoichiometry of Equation (1).−, while the reduction of Ru3+ requires H2. H2 is added to the feed in order to reduce Ru and remove another Cl− ligand. HCl is detected in the MS, suggesting the reaction between H2 and the Cl− ligand . These results match the FTIR spectrum of molecular cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] cis(CO)‐cis(Cl)‐[RuII(bipy)(CO)2Cl2] .− via CO with the help of H2 ‐trans(Cl)‐[Ru(bipy)(CO)2Cl2], showing similar Ru−Cl bond length (2.339 Å) and a slightly longer Ru−CO bond length .A further increase in the temperature does not result in any significant changes in the online MS profile, in situ DRIFTS and XAFS spectra ‐trans(Cl)‐[Ru(bipy)(CO)2Cl2] sites, feature A disappears, and a new feature C appears at 2843.0 eV. The absence of feature A suggests completely filled RuII 4d t2g6 orbitals. Thus, the reduction from RuIII to RuII is confirmed. The additional feature C is assigned to Ru 2p3/2→CO π* transition.− and cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] sites in the Cl K‐edge XAFS, representing the Cl 1s→Ru 4d eg transition ‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] site. This feature originates from Cl 1s to unfulfilled RuIII 4d t2g. Therefore, the reduction is further verified via Cl K‐edge XAFS. It also suggests that the Cl is not an outer shell counter anion but is in a close coordination environment with Ru cation. site is achieved.Molecular O2 from 503 K ‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] is active for CO oxidation. We perform CO oxidation for both mer(Cl)‐[bipy‐RuIII(CO)Cl3] and cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] sites. The former shows no conversion, whereas the latter gives a light off at 388 K and 35 % conversion at 453 K, pCO=0.01 bar and weight hourly space velocity (WHSV) of 60,000 mL h−1 g−1 . An activation energy (Ea) of 90 kJ mol−1 is determined at the WHSV of 600 000 mL h−1 g−1. Based on the EXAFS study of the cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] site and its narrow full width at half maximum (FWHM) of CO absorption in FTIR spectra , we assume that all the RuII sites are identical and perform the same in CO oxidation. The corresponding TOFs are then calculated as 0.73×10−2 s−1, 1.77×10−2 s−1, and 3.89×10−2 s−1 at 462, 479 and 498 K, respectively. The TOF and Ea are similar to the state‐of‐the‐art Cu and Pt single site over metal oxide support for CO oxidation . HAADF‐STEM images show the high density of Ru2+ cations well dispersed on the polymer framework after catalysis for cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] site . There is no sign of aggregation or cluster formation, which is further proved via ex situ XRD studies . The RuII retains the cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] structure, as indicated in the XAFS  and FTIR  studies after catalysis. The PPhen‐bipy polymer remains unchanged, according to the post catalysis FTIR and scanning electron microscopy analysis . This is because of its high stability in air at elevated temperatures . The activity of cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] is stable during the 24 h on stream stability test . The distinct differences in CO oxidation activity between mer(Cl)‐[bipy‐RuIII(CO)Cl3] single site and cis(CO)‐trans(Cl)‐[RuII(bipy)(CO)2Cl2] single site are resulted from their geometric and electronic properties. Geometrically, the former has three Cl− ligands, which are strong binding ligands in metal complexes  and leaves only one CO ligand to be exchanged during catalysis. In comparison, the latter offers two exchangeable CO ligands in cis configuration, enabling the local activation of CO within the Ru coordination environment. Electronically, the RuII is in a reduced form, which is reported to activate CO in water‐gas‐shift reaction.Further increasing the temperature shows the release of COK Figure . This suIIIBr4]− single site, where Br− is a better leaving group than Cl− according to solution based coordination chemistry. The transition from [bipy‐RuIIIBr4]− to [bipy‐RuIII(CO)Br3] takes place at room temperature while the reduction from [bipy‐RuIII(CO)Br3] to [bipy‐RuII(CO)2Br2] requires 513 K . This is 40 K higher than the temperature required to reduce the Cl coordinated Ru single sites. Without the presence of solvent, the bond breaking and formation here at the solid/gas interface are different.The role of the halogen ligand is further studied via [bipy‐Run] site is predictable according to coordination chemistry. We further extend the scope of such system to twelve metal cations based on their positions in the periodic table and their applications in catalysis. They are 3d metals, including Fe3+, Co2+, Ni2+, Cu2+, and Zn2+, noble metals, including Rh3+, Pd2+, Ag+, Ir3+, Pt4+, and Au3+, and p‐block element Sn4+. This selection covers cations with valence electrons from d5 to d10 and ionic radius from 69 pm to 129 pm, which can form tetrahedral, square planar and octahedral geometries that lead to different catalytic behaviours. [bipy‐FeCl4]−, [bipy‐CoCl2], [bipy‐CuCl2], [bipy‐ZnCl2], [bipy‐RhCl4]−, [bipy‐PdCl2], and [bipy‐PtCl4]− are obtained with good metal dispersion over PPhen‐bipy, as determined in HAADF‐STEM and XAFS studies 4]2+ sites instead of [bipy‐NiCl4]2− . Ag, Sn, Ir, and Au form a majority of clusters instead of single sites 4]2+ are achieved accordingly . The diverse selection of metal cations and ligands provides a single‐site palette with controllable geometric and electronic structures for a target catalytic reaction.The homogeneity of the single sites is crucial to provide a key platform to investigate the structure–activity relationship.s Figure –h. XANES− Figure . 4d and s Figure –k, which4]− and [bipy‐CuCl2] sites are studied in the CO oxidation under the same condition to that of the Ru single sites. Both show very limited CO conversion , despite that Pt and Cu single sites over metal oxide support are active in this reaction . Such a difference between molecularly designed Ru and Cu/Pt sites can shed light on the future research.[bipy‐PtClcis(CO)‐trans(Cl)‐[bipy‐RuII(CO)2Cl2] single site over a PPhen‐bipy framework with molecular‐level accuracy. Compared to the completely inert RuIII site, the RuII site is active for CO oxidation owing to its unique electronic and geometric structure. Such a RuII single site can only be formed in the simultaneous RuIII to RuII reduction by H2 and the ligand exchange from Cl− to CO. With the help of a well‐defined pristine single site, such solid/gas interface coordination chemistry can be used to explain the surface bond breaking and formation at molecular level. The design method is generally applicable and is versatile for metal centers that are ubiquitous in catalysis, as supported by the synthesis of [bipy‐MXn] sites for another nine elements.We have designed an air‐stable The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Inherited retinal diseases (IRDs), which are among the most common genetic diseases in humans, define a clinically and genetically heterogeneous group of disorders. Over 80 forms of syndromic IRDs have been described. Approximately 200 genes are associated with these syndromes. The majority of syndromic IRDs are recessively inherited and rare. Many, although not all, syndromic IRDs can be classified into one of two major disease groups: inborn errors of metabolism and ciliopathies. Besides the retina, the systems and organs most commonly involved in syndromic IRDs are the central nervous system, ophthalmic extra-retinal tissues, ear, skeleton, kidney and the cardiovascular system. Due to the high degree of phenotypic variability and phenotypic overlap found in syndromic IRDs, correct diagnosis based on phenotypic features alone may be challenging and sometimes misleading. Therefore, genetic testing has become the benchmark for the diagnosis and management of patients with these conditions, as it complements the clinical findings and facilitates an accurate clinical diagnosis and treatment. The retina is a multi-layered sensory tissue that lines the back of the eye. Its main function is the transduction of light energy into an electrical potential change, via a process known as phototransduction. The light-sensitive elements of the retina are the photoreceptor cells. The retina contains two types of photoreceptors, rods and cones. Rods (approximately 120 million in the human eye) are in charge of night vision, while cones (6 to 7 million in the human eye) are in charge of visual acuity and color vision. The highest cone concentration is found in the central region of the retina, known as the macula. Photoreceptors are highly compartmentalized cells, with the nucleus and other cellular organs located in the inner segment (IS), while the entire phototransduction machinery is included in the outer segment (OS).Inherited retinal diseases (IRDs), which are among the most common genetic diseases in humans, define a clinically heterogeneous group of disorders, which cause visual loss due to improper development, dysfunction or premature death of the retinal photoreceptors . IRDs arhttps://sph.uth.edu/retnet/). It can be inherited as autosomal recessive (AR), autosomal dominant (AD) or X-linked (XL). Mitochondrial and digenic modes of inheritance have also been described. While in most cases of IRD the disease is limited to the eye (non-syndromic), over 80 forms of syndromic IRD have been described. Approximately 200 genes are associated with these syndromes . We discuss different aspects, including the marked genetic heterogeneity of some of these syndromes, phenotypic overlap and diagnostic approaches.The topic of systemic diseases associated with IRDs has been reviewed before, including the description of some of these syndromes . In the The majority of syndromic IRDs are recessively inherited and rare. Many, although not all, syndromic IRDs can be classified into one of two major disease groups: inborn errors of metabolism (IEM) and ciliopathies.IEMs are genetic disorders leading to failure of carbohydrate metabolism, protein metabolism, fatty acid oxidation or glycogen storage. Many IEMs present with neurologic symptoms . The retCiliopathies are a group of genetic diseases caused by mutations in genes associated with the structure and function of primary cilia. Primary cilia function as signaling hubs that sense environmental cues and are pivotal for organ development and function, and for tissue homeostasis. By their nature, cilia defects are usually pleiotropic, affecting more than one system . PhotoreOver 80 forms of syndromic IRD have been described . Most ofBBS  is characterized by a combination of RP, postaxial polydactyly , hypogonadism, renal disease, intellectual disability (ID) and truncal obesity . Twenty-JBTS  is characterized by a peculiar midbrain–hindbrain malformation, known as the molar tooth sign. The neurological presentation of JBTS includes hypotonia that evolves into ataxia, developmental delay, abnormal eye movements and neonatal breathing abnormalities. This picture is often associated with variable multiorgan involvement, mainly of the retina, kidney and liver . RD has USH  is characterized by the combination of RP and sensorineural hearing loss (HL). Based on the severity and progression of HL, age at onset of RP and the presence or absence of vestibular impairment, the majority of USH cases can be classified into one of three clinical subtypes (USH1-3). Eleven USH genes have been identified to date (OMIM) . Their pWhen referring to syndromic IRD, phenotypic overlap is a common phenomenon, which can be divided into three groups, as detailed below:Many types of syndromic IRD have a multi-systemic nature. Certain organs are commonly involved in syndromic IRDs. Specifically, CNS involvement  is found in 68% of IRD syndromes , and oveUSH2A gene. Mutations in this gene are the most common cause of USH, and specifically of USH2  [USH2A variants are also one of the commonest causes of AR non-syndromic RP worldwide [USH2A variants determines whether one has USH2 or non-syndromic RP [USH2A-related USH2 than in patients with USH2A-related non-syndromic RP. However, the reason is not completely understood [Twenty-eight of the genes listed in ural HL) . Moreoveorldwide ,35,36. Iromic RP ,38,39. IIRD is one of the most genetically heterogeneous groups of disorders in humans, and most cases of IRD are non-syndromic. Non-syndromic IRD may coincide with other genetic (and non-genetic) rare conditions, leading to a clinical suspicion or diagnosis of a syndrome. For example, co-occurrence of non-syndromic RP and non-syndromic HL in a family may appear as USH .Due to the high degree of phenotypic variability and phenotypic overlap found in syndromic IRD, as described above, correct diagnosis based on phenotypic features alone may be challenging and sometimes misleading. Therefore, genetic testing has become the benchmark for the diagnosis and management of patients with these conditions, as it complements the clinical findings and facilitates an accurate clinical diagnosis. Establishing a correct diagnosis is important for both the patients and their family members, for multiple reasons: it enables the understanding of the natural history course, and the prediction of disease prognosis; it aids in tailoring correct follow-up and treatment, including potential gene-targeted therapies ; it leadThe existence of common founder mutations in certain populations allows for quick and efficient mutation screening in affected individuals, based on the relevant phenotype and ethnic background. This is performed by PCR-based DNA amplification and Sanger sequencing, or by specifically designed assays. Some examples are common USH3A-, USH1F-, ML4- and BBS2-causative mutations found in the Ashkenazi Jewish population ,74,75,76Currently, the most efficient approach for genetic diagnosis in monogenic diseases, including IRDs, is next-generation sequencing (NGS). NGS technologies facilitate the screening of the entire genome ; of all protein-coding regions ; or of protein-coding regions of pre-determined panels of genes  ,80. SincOver 80 forms of syndromic IRDs have been described, and approximately 200 causative genes identified. Due to the high degree of phenotypic variability and phenotypic overlap found in syndromic IRD, correct diagnosis based on phenotypic features alone is insufficient, and genetic testing has become the benchmark for the diagnosis and management of patients with these conditions. For most patients, molecular diagnosis should be based on NGS technologies. Currently, WES is the most popular approach for genetic analysis in patients with monogenic diseases, including IRDs. However, the continuous progress in both technical and bioinformatic aspects, as well as the reduction of costs, is already leading to a shift towards WGS as the method of choice.


The aim of the study was to compare the accuracy of percutaneous pedicle screw (PPS) insertion (P-side) with that of conventional open screw insertion (O-side) during unilateral open transforaminal lumbar interbody fusion (TLIF) in the same patients. We also sought to determine the incidence of pedicle screw misplacement and to identify relevant risk factors.The study was a retrospective analysis of prospectively collected data for 766 pedicle screws placed in 181 consecutive patients who underwent a unilateral open-TLIF procedure in the lumbosacral spine. Our minimally invasive TLIF was performed by unilateral open freehand insertion of pedicle screws for decompression on one side and PPS on the opposite side. Using this approach, we were able to compare the accuracy of PPS insertion with that of conventional open screw insertion in the same patients. There were 383 PPSs and 383 screws inserted by the open method. The accuracy of screw placement was evaluated on reconstructed computed tomography images obtained postoperatively, and screw misplacement was classified. Potential risk factors for screw misplacement were investigated in three-level mixed-effects logistic regression analysis.P = 0.803). Subclassification analysis revealed minor perforation of 28 screws (7.3%) on the P-side and 32 (8.4%) on the O-side, moderate perforation of 5 screws (1.3%) on the P-side and 4 (1.0%) on the O-side, and severe perforation of 1 screw (0.3%) on each side. Three-level mixed-effects logistic regression analysis identified body mass index as a significant risk factor for screw misplacement on the P-side .Thirty-four screws (8.9%) were misplaced on the P-side and 37 (9.5%) were misplaced on the O-side; the difference was not statistically significant (Accuracy of pedicle screw insertion was not significantly different between PPS insertion and conventional open screw insertion in the same patients. Body mass index had a significant influence on the risk of screw misplacement in PPS insertion. Minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) has been well described in the literature –4 and isAt our institution, we perform unilateral open TLIF using unilateral open freehand insertion of pedicle screws for decompression on one side and PPS insertion on the contralateral side. This aim of this study was to compare the accuracy of PPS insertion with that of conventional open insertion in the same patients. We also sought to determine the incidence of pedicle screw misplacement and to identify relevant risk factors.n = 160) or double-level (n = 21) unilateral open-TLIF for lumbar spinal canal stenosis with degenerative spondylolisthesis and degenerative instability between April 2011 and March 2016 at Yokohama City Minato Red Cross Hospital. Mean body mass index was 24.1 ± 3.7. The patients were evaluated preoperatively by magnetic resonance imaging (MRI) and computed tomography (CT). No patient was excluded. A total of 766 pedicle screws placed in the lumbosacral spine, of which 383 were PPS (P-side) and 383 were pedicle screws inserted by the conventional open method (O-side). Two screws were placed at L2, 47 at L3, 141 at L4, 145 at L5, and 48 at S1  who underwent single-level .The patient was placed in the prone position with the trunk on a Relton-Hall frame. Following induction of anesthesia, standard surgical exposure was performed, with a 5-cm midline skin incision and unilateral exposure of the transverse processes. The open side was decided based on the patient’s symptoms. The base of the transverse process was perforated using an air drill, and marking wires  were inserted into the pedicle. After the positions of the marking wires were confirmed using C-arm fluoroscopy, the wires were removed and the pedicles were tapped using the freehand technique. Pedicle screws were then inserted into the pedicles using the freehand method. We performed unilateral laminectomy, bilateral decompression for central stenosis, and foraminal decompression for foraminal stenosis using microscopy. After decompression, bone grafts and 2 cages were packed into the disc space. The wound was closed in layers Fig. .Fig. 1PNext, a 1.5-cm stab incision was made laterally on the side opposite to the open side. The targeting needle was then inserted into the pedicle at the superolateral border based on the anterior-posterior view under C-arm fluoroscopic guidance. The guidewire was inserted through the targeting device and into the pedicle. The pedicle was tapped using the guidewire. Finally, the pedicle screw was inserted over the guidewire. Other PPSs were inserted using the same method Fig. . The sizPostoperative CT scans were obtained using a 16-row multidetector CT system  to assess the implant position in all patients. The series consisted of 3.0-mm CT sections that were reconstructed at 2.0-mm intervals. A spine surgeon not otherwise involved in the study evaluated the position of the screw in the pedicle wall in the axial, sagittal, and coronal planes. Screw misplacement was defined using the system proposed by Schizas et al.  as minorU test. To identify risk factors for misplacement, three-level mixed-effects logistic regression analysis was performed that accounted for possible clustering of screws (level 1) nested within the vertebral level (level 2), and the vertebral nested within the individual (level-3). We adjusted for age, sex, and BMI and the operated side and level. Statistical analysis were performed using the STATA version 16.0  and the commercial package JMP Version 13.1.0 software . A P-value < 0.05 was considered statistically significant.Statistical analysis was performed using Fisher’s exact test and the Mann-Whitney P = 0.803). Subclassification analysis revealed minor perforation of 28 screws (7.3%) on the P-side and 32 (8.4%) on the O-side; moderate perforation of 5 screws (1.3%) on the P-side and 4 (1.0%) on the O-side, and severe perforation of 1 screw (0.3%) on each side. The direction of misplacement was inferior for 2 screws on the P-side and 1 screw on the O-side, superior for 2 screws on the P-side and 1 screw on the O-side, medial for 22 screws on the P-side and 23 screws on the O-side, and lateral for 8 screws on the P-side and 12 screws on the O-side. Screw misplacement occurred at the following vertebral levels: L2 for 1 screw on the P-side and 1 screw on the O-side, L3 for 3 screws on the P-side and 5 screws on the O-side, L4 for 18 screws on the P-side and 15 screws on the O-side, L5 for 8 screws on the P-side and 15 screws on the O-side, and S1 for 4 screws on the P-side and 1 screw on the O-side. Four screws on the P-side and 2 screws on the O-side showed moderate or severe penetration medially. One screw on the P-side and 1 screw on the O-side were associated with neurological symptoms and therefore were replaced in both cases  were inserted in 181 patients. The number of successfully placed pedicle screws was 349 (91.1%) on the P-side and 346 (90.5%) on the O-side. Screw misplacement was observed for 34 screws (8.9%) on the P-side and 37 (9.5%) on the O-side; the difference was not statistically significant , likely because bone  becomes more fragile with aging and is more easily perforated. Furthermore, Oh et al. reported that bone density was an important factor in pedicle wall penetration. The penetration rate was higher in patients with stronger bones in whom the direction of screw insertion was difficult to modify. However, the trajectory can easily be changed during the procedure in weak bone [In our study, older age tended to be a risk factor when using conventional open insertion (eak bone . It has eak bone . We beliThis study has several limitations. First, some of the insertion procedures were performed by spine surgeons and others by residents. Second, the study had a retrospective single-center design. However, the position of the screw was assessed by a spine surgeon who was not otherwise involved in the study and a 16-row multidetector CT system was used to confirm the implant position in all patients.This is the first study to compare the accuracy of pedicle screw insertion between PPS insertion and conventional open insertion in the same patients. There was no significant difference in accuracy between the two techniques. BMI was found to be a significant risk factor for screw deviation with PPS insertion but not with conventional open insertion. These findings suggest that the accuracy of PPS insertion is affected by BMI because this method relies heavily on radiologic imaging and that PPS insertion is thus not optimal in obese patients.


Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a binding affinity approximately seven times lower than NKp46 binding of influenza A’s HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that NKp46 is not important for influenza B elimination. These findings enable us to better understand the interactions between the different influenza viruses and NK cells that are known to be crucial for viral elimination. Natural Killer (NK) cells are lymphocytes that are part of the innate immune system . They arInfluenza infections can lead to hospitalization and death among high-risk individuals , thus posing major health and economic burdens. Part of IAV’s deadly nature is its ability to change at a high frequency the virus’ membrane proteins, hemagglutinin (HA) and neuraminidase (NA) , a phenoInfluenza B (IBV) is somewhat less common than IAV and almost exclusively infects humans. The only two known lineages of IBV were discovered in the late 1970s and are named Victoria and Yamagata . DespiteHere we show that NKp46 recognizes IBV via binding to HA. We discovered the mechanism of binding and identified the binding site on NKp46 that is crucial for its interaction with IBV’s HA. We found that the affinity of NKp46 to the HA of IBV is significantly weaker than to that of IAV and that NKp46 is not involved in the clearance of IBV in vivo.The cell lines used in this study were the human choriocarcinoma cell line JEG-3 and the murine lymphoma cell line EL4 (ATCC). Human peripheral blood lymphocytes were separated from buffy coats using centrifugation on Ficoll gradient (Lymphoprep) followed by NK cells’ isolation using the Easy Sep Negative selection human NK cells enrichment kit (StemCell Technologies) according to manufacturer’s protocol. Mouse NK cells were isolated from the immune competent Ncr1+/gfp and from the NCR1-deficient Ncr1gfp/gfp mice using the Easy Sep Negative selection mouse NK cells’ enrichment kit (StemCell Technologies) 18 h after intraperitoneal injection of 200 μg poly(I):poly(C) (Sigma). The human influenza viruses A/Brisbane/59/2007 H1N1 and B/Brisbane/60/2008 (Victoria lineage) used in this study were generated as previously described [Monoclonal antibodies (mAbs) used in the present study included anti-HA specific to A/Brisbane/59/2007 and anti-HA specific to B/Brisbane/60/2008 that we kindly received from the International Reagent Resource (IRR) that was established by the Center for Disease Control and Prevention (CDC). The hNKp46.02 antibody was previously described . The antCells were coated overnight with 20 μL of A/Brisbane/59/2007 H1N1 or B/Brisbane/60/2008 at 37 °C. The cells were then washed and incubated with the appropriate antibody (0.2 μg/well) or fusion protein (0.5 μg to 5 μg/well). Then, cells were stained and analyzed using fluorescence-activated cell sorting (FACS). All results were analyzed using the FCSExpress 6 software.The various recombinant HA proteins mentioned above were coated to an ELISA plate (0.1 μg/well). Blocking was performed with BSA 1%. 0.1 μg/well of NKp46-Ig or anti-HIS-Tag  were added followed by the relevant biotinylated secondary antibody from Jackson ImmunoResearch. The plate was then incubated with Streptavidin HRP . Binding was measured by using TMB solution and reading of the results was done with an ELISA reader at a wavelength of 650 nm. The binding of NKp46 to the different HA proteins was normalized to the amount of HA that was bound to the well, which we determined using the anti HIS-tag antibody.We used HIS capture kit to coat the different recombinant HA proteins to a CM5 chip . After coating of the HA, we performed single cycle kinetics, as described before , by push35S labeled methionine. In the same time, human activated NK cells were incubated with hNKp46.02 for the internalization assay, as previously described [35S release from lysed cells, as previously described [Targets with or without influenza were incubated overnight with radioactive escribed . The nexescribed .All animal work was conducted according to relevant national and international guidelines. The work was approved by the Hebrew University Medical School Ethic committee . All experiments were performed using 6–8- weeks-old females of C57BL/6 background. The generation of the Ncr1gfp/gfp mice was described before . Mice wep < 0.05. ANOVA was used to identify significant group differences See figure legend for further detail.GraphPad Prism software version 8.0 was used for all statistical analyses. For statistical significance, two-tailed Student’s t test analysis was used. A statistical test was considered significant (*) when To test whether NK cells recognize IBV, we incubated Jeg3 cells with IAV or IBV. Jeg3 cells express very few NK activating ligands and are therefore not killed well by human NK cells, making them ideal candidates for testing the interaction between NK cells and the abovementioned viruses . For IAVWe previously demonstrated that the viral ligand of IAV recognized by NKp46 is the HA protein . As can Since NKp46 binds the HA of IBV with lower affinity than IAV, we next tested whether the recognition of IBV by NKp46 is different from that of IAV. We previously showed that the binding of NKp46 to IAV is dependent on sialic acids present on NKp46 . To testThe specific glycosylated residue critical for the binding of NKp46 to IAV was previously identified to be threonine 225 (Thr 225) . To testTo investigate the functional implications of NKp46 binding to IBV we used an anti-NKp46 mAb, named hNKp46.02, that we previously generated. This specific antibody, when incubated with cells expressing NKp46, leads to internalization and degradation of the receptor; therefore, NKp46 is downregulated from the cell surface .35S-labeled Jeg3 cells in the presence or absence of IBV and a cytotoxicity assay was performed. In the absence of IBV, Jeg3 cells were weakly killed and the hNKp46.02 antibody reduced the killing even further , mice or from the Ncr1 deficient, Ncr1gfp/gfp (KO), mice that we previously generated . In the We incubated the NK cells obtained from the two strains of mice with EL4 in the presence or absence of IAV and IBV. As expected, and in agreement with previous results , when weTo determine the physiological relevance of our findings we injected mice intranasally with IAV or IBV. Five days post infection we sacrificed the mice and assessed viral load in the lungs. As previously reported , when weInfluenza infections constitute a major health and economic burden . AccordiInterestingly, it was not known whether NK cells recognize IBV. Influenza B viruses diverged from influenza A viruses at some point in the distant past and are, therefore, newer viruses . This viHere we showed that NKp46 binds to the HA of IBV mainly through sialic acids present on the threonine 225 of NKp46. This mode of binding is similar to that of IAV . InteresNext, we demonstrated that NKp46 has a lower affinity against the HA of IBV as compared to IAV. Nevertheless, the NK cells were still able to kill cells in the presence of IBV, and this was dependent on NKp46. Interestingly, when assessing whether mouse NK cells were able to kill cells in the presence of IBV, we observed that, although NCR1 is able to bind IBV, this interaction does not induce killing. In this assay both IAV and IBV were evaluated, and mouse NK cells expressing NCR1 were able to efficiently kill cells in the presence of IAV but not IBV. This observation suggests that NCR1 may not play a role in the clearance of IBV. We confirmed this assessment using an in vivo system where we observed that the presence or absence of NCR1 did not influence viral clearance of IBV. It is possible that the lower affinity of NKp46 to IBV HA is because the HA protein of IBV binds α-2,3 as well as α-2,6-linked sialic acids. The sialic acids present on the threonine 225 of NKp46 are mainly α-2,6-linked sialic acids . Moreove