Acenes and azaacenes lie at the core of molecular materials’ applications due to their important optical and electronic features. A critical aspect is provided by their heteroatom multiplicity, which can strongly affect their properties. Here we report pyrazinacenes containing the dihydro-decaazapentacene and dihydro-octaazatetracene chromophores and compare their properties/functions as a model case at an oxidizing metal substrate. We find a distinguished, oxidation-state-dependent conformational adaptation and self-assembly behaviour and discuss the analogies and differences of planar benzo-substituted decaazapentacene and octaazatetracene forms. Our broad experimental and theoretical study reveals that decaazapentacene is stable against oxidation but unstable against reduction, which is in contrast to pentacene, its C–H only analogue. Decaazapentacenes studied here combine a planar molecular backbone with conformationally flexible substituents. They provide a rich model case to understand the properties of a redox-switchable π-electronic system in solution and at interfaces. Pyrazinacenes represent an unusual class of redox-active chromophores. Acenes are a promising class of organic materials with distinctive electronic and optical properties. Here decaazapentacene and octaazatetracenes are prepared and their self-assembly behaviour at different oxidation states is analysed. An increasingly important challenge is to control site-specific redox functionality and physicochemical properties of molecules within supramolecular architectures5, especially at surfaces and interfaces10 where access to such functionality and the corresponding applications would be facilitated even at the molecule level12. Biochemical systems have evolved towards exceptional levels of control over the supramolecular and metal coordination mechanisms governing chemical reactions13. These structures, however, are usually too delicate for technological applications. On the other hand, the engineering of organic interfaces and electrochemically or optically functional surfaces also requires precise control over the assembly of redox functional systems on surfaces14. Thus, established structure–function relationships of molecular materials known from solution need to be re-evaluated to predict and understand the interface-specific chemical, electronic, optical and mechanical properties of any newly synthesized molecules. Along these lines, we here discuss a special class of heteroacenes in-solution and on surfaces, in particular for their complex and interesting redox activity and supramolecular chemistry by taking a broad experimental approach in conjunction with ab initio property prediction techniques.Redox chemistry, i.e. the controlled oxidation/reduction of atoms, also within molecules affecting their chemical, electronic and optical properties, has been a significant driver e.g. for synthetic and materials chemistry18, the pyrazinacenes21, composed of linearly fused pyrazine rings, possessing multi-stability of redox/photo-redox chemistry in-solution and when adsorbed at a Cu(111) surface. The physicochemical properties of these molecules are discussed in the context of their modified redox state before and after on-surface reactions: we link their different observed oxidation states to their conformation, their configurations and their aggregation on surfaces. The complexity of this system derives from its molecular architecture, which combines a planar molecular backbone with substituents exhibiting conformational freedom and a redox-variable π-electronic system. The first step of oxidation enables cooperative conformational adaptability and aggregation. The second step leads to chemisorption prohibiting self-assembly of the molecules due to strong interactions with the substrate. Indeed, it is remarkable that a redox-active molecular module can be made that does not polymerize by reaction or coordination on surfaces but rather expresses the same conformational states in-solution and on surfaces also involving assembly into 2D islands. The non-planar molecular morphology together with the three-state redox system are key to the complexity observed and have been confirmed independently using high-resolution scanning tunneling microscopy (STM) and ab initio density functional theory (DFT) simulations (see Supplementary Information (SI) for experimental and computational details).We report N-heteroacenes21. The native and oxidized forms are chemically highly stable and do not undergo auto-catalysed or surface-catalysed reactions. Similar systems can be built/designed, in particular if larger ‘protective’ substituents can be incorporated into less inert multi-stage redox systems as seen in protein biochemistry where the bulky polypeptide backbone protects redox centres from prevailing solution conditions and defines its selectivity22. Delocalized electron systems comprising N and C π-electronic states24 qualify the pyrazinacenes for the investigation of their light absorption as a chromophore and their redox behaviour, also within specific supramolecular and on-surface architectures.The pyrazinacenes used here are stable, length-tunable redox-active molecular modules, which can be activated in three steps. Remarkably, pyrazinacenes are sufficiently stable at each stage of oxidation in different media and in different states of aggregation29. Decaazapentacenes  (vide infra).Pyrazinacene-type molecules bearing N-substituents have been prepared previously and are known to be highly fluorescent molecules with potential applications beyond the usual acene-type applicationsro-3 see . Initial30. 1 and 2, due to their similar structures, appear also similar when studied by STM on a Cu(111) surface, which we have used as a model substrate to investigate their supramolecular self-assembly and reactivity at the interface, as it evolves with progressive chemical conversion. Details of 2 are shown  to their native oxidation state containing one reduced pyrazine ring.The initial impetus for this work was to study pyrazinacenes at metal surfaces in particular to observe the effects of the dihydropyrazine unit, which is known to be formally electronically delocalized yielding a planar molecular morphologyogy Fig. . The alm2. These XPS energy values are highly consistent with those previously observed for similar enamine NH and pyridinic N atoms whose signals appear, respectively, at 399.632 and 398.3 eV32.The molecules are adsorbed in registry with the substrate with 6 different orientations  is present in molecules contained in the line structure indicating that dehydrogenation of the pyrazinacene core of 2 has occurred yielding what we assign as 2-ox. Annealing at 150 °C appears to increase the rate of line formation but also leads to the appearance of unusual double-lobed structures different from the starting morphology. To investigate this, we annealed at higher temperature  is also known to act as a cyclodehydrogenating agent at elevated temperatures33 such that 2-ox is converted to 2-ox2 containing terminal phenanthrene units and re-modifying the charge transfer and on-surface self-assembly. The STM profiles of 1 and 2  surface. Notably this reaction yields tetrabenzo-5,7,9,11,18,20,22,24-octaazahexacene whose dimensions and profile fit well with those observed. There is also no substantial variation in the XPS spectra  (whose non-co-planar phenyl substituents remain visible), which assembles to the line structure, followed by cyclodehydrogenation to 2-ox2 (1-ox2), a highly planar pyrazinacene species, which does not self-assemble, resulting in elimination of the 2-ox (1-ox) line structure.As summarized in Fig. 1 and 2 behave similarly. As shown in Fig. 2, 2-ox and 2-ox2,  interaction with the metal substrate. In contrast, in 2-ox the molecular backbone is twisted to accommodate interactions between the substrate and the pyridinic N electron pairs on the acene. Note that acenes are known to be able to accommodate significant twisting of the backbone34. Thus, the modification of the interaction of the nitrogen atoms is plausibly weak and essentially undetectable by XPS in spite of a slight broadening . 1 shows an acene-type 4-banded spectrum with an absorption maximum at 500 nm and shorter wavelength peaks diminishing in intensity. 2 has a similar several banded spectrum except that the band at 450 nm (the 2nd acene band) is the absorption maximum; a feature we attribute to tautomerization in this molecule and which has been observed for fluoflavine 36 earlier. 1-ox and 2-ox were obtained by treating solutions of 1 or 2 with PbO237 both undergoing a shift in absorption maximum to longer wavelength  = 0.64, ΦF(2) = 0.31) or acid  = 0.26, ΦF(2) = 0.15) revealing that these compounds could be sensitive reversible probes of acidity/basicity or redox conditions based on changes in their fluorescence emission.The compounds ved Fig. . Quantum1 and 2 can also be achieved electrochemically  with the distinct blue colour of 1-ox emerging during heating at temperatures above 400 °C . Heating either of the compounds leads to colour changes to those of their oxidized states (accompanied by UV-vis changes consistent with those). TGA of 1 indicates the excellent stability of this compound  up to over 400 °C and supports the assignment of 1-ox in the on-surface studies.Oxidation of see Fig.  with 2 b1-ox and 2-ox are sufficiently stable to allow the measurement of their UV/Vis spectra  revealing the relative stability even of their anions in the solution state  mirrors the relative instability of pentacene. This might be related to the electron deficiency of DAP and indicates that special treatment (i.e. to exclude reducing species) will be required in investigations of the properties of these oxidized states. Photolytic redox transformations of 1 and 2 also suggest their potential as photocatalysts   and dihydro-decaazapentacene (1), i.e. the 8- and 10-nitrogen atom-substituted analogues of tetracene and pentacene, have been selected to represent these acenes. They are shown to be oxidized. respectively. to octaazatetracene (2-ox) and decaazapentacene (1-ox) both in-solution and in situ on a solution-free surface in a vacuum in a scanning tunneling microscope. The oxidized molecules aggregate with specific supramolecular chain motifs. Onward temperature-induced cyclodehydrogenation of their phenyl substituents creates their planar analogues, 1-ox2 and 2-ox2, respectively. These adsorb as individual molecules exhibiting distinct conformations indicating that oxidation state has a critical effect on the supramolecular polymerization in this system. Our electrochemical data supports the multi-stage ionization and relatively low first oxidation potentials . In photo-stimulated redox reactions, C–C bonds are formed catalytically. For their easy dehydrogenation, in particular at interfaces, we propose that these compounds may be useful as electron/proton transfer conduits for catalysis. It is notable that 1 and 2 are both sufficiently stable to be used under reaction conditions involving irradiation where similar acene compounds may not be stable. The molecular analogues of 1-ox and 2-ox, pentacene and tetracene, are widely considered of most importance for their semiconductivity and their consequent use in e.g. organic electronics and optoelectronics. Decaazapentacene, on the other hand, appears to be interesting because of its photochemical behaviour as a proton-coupled chromophore that can be oxidized, deprotonated and protonated as reported here. Also, decaazapentacenes provide an example of surface-specific reactivity, i.e. of the formation of 1-ox2 and 2-ox2 exclusively on the surface, which could not be reproduced in-solution.In this unique side-by-side investigation using different complementary techniques, we report the synthesis, the physicochemical properties and in particular the oxidation-state-dependent self-assembly of pyrazinacenes. Dihydro-octaazatetracene  using a Hamamatsu Photonics C9920-02G Quantum Yield Spectrophotometer. Fourier transform infrared spectra were obtained from samples deposited on a barium fluoride disc using a Thermo-Nicolet 760X FT-IR spectrophotometer. Proton and carbon-13 nuclear magnetic resonance (1H-NMR and 13C NMR) spectra were obtained using a JEOL AL300BX spectrometer  or a Bruker Avance III 600 MHz spectrometer  with tetramethylsilane (TMS) as an internal standard. MALDI-TOF mass spectra were obtained using a Shimadzu Axima AFR+ mass spectrometer using dithranol as the matrix. High-resolution (HR) MALDI-TOF mass spectra were measured using an LTQ Orbitrap XL system using 2,5-dihydroxybenzoic acid as matrix. Computational geometry optimizations were performed using NWChem43 at the B3LYP/6-311 + G level. GaussView (in GAUSSIAN44) was used to generate images of frontier HOMO and LUMO orbitals. Cyclic voltammograms were recorded on an EG&G Model 263 A potentiostat operating a three-electrode system. The working electrode was a platinum button with a platinum wire serving as the counter electrode. An Ag/AgCl electrode was used as the reference. Tetra-n-butylammonium perchlorate (0.2 M) was used as the electrolyte and voltammograms were recorded at a scan rate of 100 mV s−1. Ferrocene/ferrocenium redox couple was used as internal standard and all solutions were purged using argon gas prior to electrochemical/spectral measurements. Spectroelectrochemical study was performed by using a cell assembly (SEC-C) supplied by ALS Co., Ltd. . This assembly consists of a Pt counter electrode, a 6 mm Pt gauze working electrode, and an Ag/AgCl reference electrode in a 1.0 mm path length quartz cell. Optical transmission was limited to 6 mm covering the Pt gauze working electrode. 5,6-Diphenyl-2,3-diaminopyrazine (6) was prepared according to a reported procedure45. Syntheses of 2,3-dichloro-6,7-diphenyl-1,4,5,8-tetraazanaphthalene, 2,3-diamino-6,7-diphenyl-1,4,5,8-tetraazanaphthalene, 2,3-dichloro-6,7-di(4-t-butylphenyl)-1,4,5,8-tetraazanaphthalene and 2,3-diamino-6,7-di(4-tert-butylphenyl)-1,4,5,8-tetraazanaphthalene are given in Supplementary Methods in the Supplementary Information.Reagents and dehydrated solvents used for synthesis and spectroscopic measurements were obtained from Aldrich Chemical Co., Tokyo Kasei Chemical Co., Wako Chemical Co. or and were used as received. Electronic absorption spectra were measured using JASCO V-570 UV/Vis/NIR spectrophotometer, Princeton Applied Research (PAR) diode array rapid scanning spectrometer or a Shimadzu UV/Visible spectrophotometer. Fluorescence spectra were measured using a JASCO FP-670 spectrofluorimeter. Fluorescence quantum yields were measured from solutions of the compounds in dichloromethane  in DMSO (5 mL) was heated for 3 h at 110 °C. The reaction mixture was allowed to cool to room temperature then poured into saturated aqueous ammonium chloride solution (30 mL) and extracted with chloroform (3 × 50 mL). The combined extracts were washed with brine (100 mL), dried over anhydrous Na2SO4 then solvents evaporated under reduced pressure. The product was isolated using column chromatography  to give the product as an orange crystalline solid. Yield: 70 mg (43 %). 1H-NMR  δ = 7.14–7.22 , 7.33–7.36 , 10.94  ppm. 13C NMR  δ = 131.3, 131.5, 133.2, 134.1, 142.3, 149.3, 152.0 ppm. FT-IR (BaF2) ν = 3639, 2956, 2924, 2854, 1522, 1445, 1429, 1406, 1368, 1185, 1088, 1026, 984, 762, 695 cm–1. HR-MS : m/z calc. for C34H22N8 [M + 2H]+: 544.21184; found 544.21385. UV-Vis (CH2Cl2): λmax (ε) = 249 (77000), 441 (39500), 461 (67000), 489 (46000) nm. Elemental analyses: calc’d for C34H24N8.2(C4H8O).1/2(C6H14): %C 74.05, %H 6.21, %N 15.35; found %C 74.51, %H 5.68, %N 15.55.A mixture of 2,3-dichloro-6,7-diphenyl-1,4,5,8-tetraazanaphthalene , 2,3-diamino-5,6-diphenylpyrazine  and K2CO3  in DMSO (5 mL) was heated for 4 h at 120 °C. The reaction mixture was allowed to cool to room temperature then poured into aqueous saturated ammonium chloride solution (30 mL) and filtered. The filtrate was dissolved in THF, the resulting solution passed through a short plug of silica then the solvents were evaporated under reduced pressure. Ethyl acetate (20 mL) was added to the resulting solid followed by ultrasonication of the suspension for 10 min. The suspension was filtered, rinsed with water (50 mL), hot ethyl acetate (10 mL), dichloromethane (10 mL) and dried under reduced pressure to give the product as a red crystalline solid. Yield: 63 mg (35 %). UV-Vis (CH2Cl2): λmax (ε) = 252 , 470 , 502  nm. 1H-NMR  δ = 7.05–7.08 , 7.15–7.17  ppm. 13C NMR  δ = 131.6, 132.2, 133.5, 135.4, 143.0, 147.9, 155.6 ppm. FT-IR (BaF2): ν = 3631, 3384, 3085, 2948, 1580, 1507, 1445, 1192, 1089, 1026, 696 cm–1. HR-MS : m/z calc. for C36H22N10 [M–H]-: 593.19561; found 593.19331. Elemental analyses: calc’d for C34H23N10.2.5H2O: %C 67.60, %H 4.25, %N 21.90; found %C 67.82, %H 4.06, %N 21.63.A mixture of 2,3-dichloro-6,7-diphenyl-1,4,5,8-tetraazanaphthalene , 2,3-diamino-6,7-diphenyl-1,4,5,8-tetraazanaphthalene  and K2CO3  in DMSO (5 mL) was heated for 4 h at 120 °C. The reaction mixture was allowed to cool to room temperature, poured into aqueous saturated ammonium chloride solution (30 mL) then the mixture extracted with CHCl3 (3 × 50 mL). The combined extracts were washed with brine (50 mL), dried over anhydrous Na2SO4 then solvents were evaporated under reduced pressure. The solid residue was purified using column chromatography  to give the product as a dark purple crystalline solid. Yield: 110 mg (45 %). UV-Vis: λmax (ε) = 231 , 268 , 571 , 606  nm. 1H-NMR : δ = 7.25–7.30 , 7.40–7.45 , 11.61  ppm. 13C NMR  δ = 31.7, 36.9, 128.5, 130.2, 131.8, 142.37, 147.3, 155.5, 160.8 ppm. FT-IR (BaF2) ν = 3213, 3129, 3051, 2960, 2924, 2855, 1607, 1542, 1442, 1286, 1190, 1116, 1077, 1016 cm−1. HR-MS : m/z calc. for C52H54N10 [M − H]−: 817.44601; found 817.44292.A mixture of 2,3-dichloro-6,7-bis(4-tert-butylphenyl)-1,4,5,8-tetraazanaphthalene , 2,3-diamino-6,7-bis(4-tert-butylphenyl)-1,4,5,8-tetraazanaphthalene  and KDihydroazaacene derivative (0.001 mmol) was dissolved in dry dichloromethane (3 mL) and lead (IV) oxide (50 wt%) was added. The reaction mixture was stirred for 10 min. then filtered through Celite and solvents evaporated under reduced pressure to give the products as solids in quantitative yields.1H-NMR : δ = 7.45–7.50 , 7.56–7.61 , 7.82–7.86  ppm. FT-IR (BaF2) ν = 3051, 2916, 2848, 1593, 1540, 1427, 1366, 1130, 768, 698, 693 cm−1. HR-MALDI-MS (DHB): m/z [M + H]+ calcd for C34H21N8: 541.1889; found: 541.1888.Dark purple solid. 2) ν = 2947, 2865, 1580, 1539, 1445, 1192, 1089, 1070, 697 cm−1. HR-MALDI-MS (DHB): m/z [M + H]- calcd for C36H21N10: 593.1951; found 593.2013. This compound is too insoluble to perform solution state NMR analyses.Dark red solid. FT-IR : δ = 1.40 , 7.52 , 7.88  ppm. 13C NMR  δ = 30.9, 35.2, 125.8, 130.9, 134.6, 148.6, 148.7, 156.8, 163.8 ppm. FT-IR (BaF2) ν = 3067, 2962, 2904, 2868, 1603, 1505, 1407, 1340, 1304, 1196, 1097, 1062, 831, 704 cm−1. HR-MALDI-MS (DHB): m/z [M + H]− calcd for C52H53N10: 817.4460; found 817.4432.Dark purple solid. 2 were grown by diffusion of hexane into a solution of 2 in tetrahydrofuran. Data collections were performed using MoKα radiation (λ = 0.71073 Å) on a RIGAKU VariMax Saturn diffractometer equipped with a CCD detector. Prior to the diffraction experiment the crystals were flash-cooled to 213 K in a stream of cold N2 gas. Cell refinements and data reductions were carried out by using the d*trek program package in the CrystalClear software suite46. The structures were solved using a dual-space algorithm method (SHELXT)47 and refined by full-matrix least squares on F2 using SHELXL-201448 in the WinGX program package49. Non-hydrogen atoms were anisotropically refined and hydrogen atoms were placed on calculated positions with temperature factors fixed at 1.2 times Ueq of the parent atoms and 1.5 times Ueq for methyl groups. Crystal data for 2: orange bar, C42H34N8O2, Mr = 682.77, monoclinic P21/n, a = 15.6972(4) Å, b = 5.71170(10) Å, c = 19.7826(6) Å, β = 106.4810(10)°, V = 1700.79(7) Å3, T = 213 K, Z = 2, Rint = 0.0428, GoF = 1.063, R1 = 0.0793, wR = 0.2500.Crystals of 1 and 2. All sample preparations were performed under ultrahigh vacuum (UHV) conditions with a base pressure of ∼10−10 mbar. The crystals were cleaned using cycles of sputtering with Ar+ ions with subsequent annealing at 450 °C. Thermal evaporation at 300 °C was used to deposit the molecules on the substrate using a commercial evaporator . The sublimed amounts of the compounds were controlled by using a quartz crystal microbalance. STM imaging was performed in constant current mode  with the selected sample bias in the range of 3 mV to 1 V at 5 K. Pt-Ir wires  were used to make scanning probe tips and these were cleaned by sputtering with Ar+ ions prior to use.All experiments were performed with an Omicron low temperature STM operating at 4.8 K in ultrahigh vacuum (UHV). A clean Cu(111) surface was prepared in situ by repeated cycles of sputtering and annealing. Mechanically sharpened and sputter-etch cleaned PtIr tips were used. Molecules were deposited from a Knudsen cell by resistively heating at about 350 °C for both XPS was used at room temperature to track the N1s chemical environment changes of the compounds before and after annealing steps. The spectra were recorded in normal emission with an instrumental setup that gives a full width at half maximum (FWHM) of 1.0 eV using a monochromatized Al Kα X-ray source.https://launchpad.net/siesta and refs. 51). This uses norm-conserving pseudopotentials52 and expands the wave functions of valence electrons in LCAO form. As exchange-correlation functional, we have used the vdW-DF-cx functional of Berland and Hyldgaard (BH)53. The geometric models were constructed based on the experimental bulk parameter for copper, 3.62 Å since the BH is known to reproduce well bulk properties of coinage metals. The systems were confined to unit cells that allow the study of periodically repeating Cu(111) surface, and included three atomic layers. The super-cell sizes vary from 7 × 10 Cu atoms for monomers to 12 × 12 Cu atoms for dimers. The length of the cells along the OZ axis was 30 Å for all systems, thus allowing a vacuum level of ~20 Å, which is large enough to avoid the artificial influence of the electric charge from one cell to another. The Monkhorst-Pack grid for the integrals in the Brillouin zone was 2 × 2 × 1. We used double-zeta polarized (DZP) basis sets with an energy shift of 100 meV for all atoms. The relaxation procedure involves the Conjugated Gradient method. The systems were relaxed by keeping the deeper layer of the copper substrate pinned at their bulk positions, only the upper layer and the molecule being permitted to relax until the maximum gradient in the structure was below 0.05 eV/A. Simulations of the STM images were performed by using the Tersoff-Hamann approximation 54). We computed LDOS in the energy window spanning from the Fermi level of the system up to 1 eV below the Fermi level, corresponding to an external bias of 1 V. We scanned the surface by searching for a given constant value of the LDOS (i.e. we simulate a “constant current” STM experiment).Simulations were performed using the Siesta code (See the Siesta distribution site, 55.Experimental and simulated STM data were processed using the WSXM softwareSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Peer Review File


The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. Influenza A viruses (IAVs) are enveloped negative-strand RNA viruses with segmented genomes containing eight gene segments. IAVs cause seasonal epidemics and occasional pandemics due to the high degree of genomic plasticity and continued evolution that enables them to escape host immunity and adapt to new host species . The IAV17–The intrinsic multifunctionality of M1 during the various steps of the IAV life cycle, together with its differential intracellular localization and multimerization state, requires its versatile regulation at multiple levels. These include differential and stage-specific protein-protein interactions, conformational flexibility, and the reversible attachment of different posttranslational modifications (PTMs) –24. Acco–25–Here, we have found that regulated M1 threonine 108 (T108) phosphorylation is required for IAV replication. Absent T108, phosphorylation prohibits its nuclear entry and M1-mediated nuclear vRNP export and leads to strongly increased self-association of M1. M1 T108 phosphorylation also controls association with host cell proteins, including members of the STRIPAK complex, which exerts a proviral function.trans (data not shown)  and by others for the H1N1-type laboratory strain A/WSN/33 (WSN) , 32. Thiown) see  and B. T10.1128/mbio.03231-22.1TABLE S1Table S1, XLSX file, 2.7 MB.Number and morphology of foci formed by SC35M WT and M1 T108 mutants. Supernatants from 293T/MDCK-II cells transfected with 8 plasmids were passaged on MDCK-II cells, followed by serial 1:10 dilutions. These were used to infect MDCK-II cells grown in 96-well plates, as schematically shown in Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03231-22.2TABLE S2Table S2, XLSX file, 2.3 MB.Number and morphology of foci formed by SC35M WT and M1 T108 mutants. Supernatants from 293T/Vero-E6 cells transfected with 8 plasmids were passaged on Vero-E6 cells, followed by serial 1:10 dilutions. These were used to infect Vero-E6 cells grown in 96-well plates, as schematically shown in Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03231-22.5FIG S1FIG S1, PDF file, 0.1 MB.Substitutions at M1 T108 did not affect the M1 protein expression levels. 293T cells were transfected to express the SC35M genome together with WT M1 or its phosphorylation-defective or phospho-mimicking versions. Extracts from cells lysed at 12 and 24 h.p.t. were further analyzed by immunoblotting for protein expression, as shown. Download Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03231-22.6FIG S2FIG S2, PDF file, 0.2 MB.Rescue experiments. (A) 293T/MDCK II cells were transfected to express the SC35M genome using bidirectional pHW2000 plasmids in an approach cotransfecting M1 WT together with M1 T108A. IAVs contained in supernatants were further characterized by titration, and virus progeny was isolated from individual plaques as shown schematically. Twelve plaques were purified, followed by amplification of the M1 gene using PCR and sequencing. (B) Results of the sequencing experiments are shown; the T108 codon is boxed. Download Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the As M1 T108 is located close to the NLS of the M1 protein (amino acids 101 to 105), a possible function of this phosphorylation for the regulation of M1 nuclear import and subsequent M1-dependent nuclear export of vRNPs was explored. In the absence of recombinant viruses expressing the M T108A mutant, 293T cells were transfected with 8 plasmids expressing all SC35M proteins including the WT M1 or its T108 phospho mutants. Indirect immunofluorescence revealed the localization of WT M1 in the cytosol and nucleus at 12 h posttransfection (h.p.t.) . Consist33–37–We then determined the relevance of M1 phosphorylation for its localization to the membrane-proximal space, where it is found at later stages of the infection , 42. ThiAssociation of M1 with the plasma membrane triggers its oligomerization as a consequence of an increased affinity to other monomers, ultimately providing structural integrity and stability to the virion –45. The –P ≤ 0.05) to M1 WT  after 24 h of protein expression , which caused the 2009 pandemic , and the viral titers were determined at the indicated time points. Bars indicate means ± SD obtained from three independent experiments performed in triplicate. **, Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03231-22.9FIG S5Strn/Strn3 or an adequate scrambled control as shown. (Left) One fraction of the cells was infected with SC35M (MOI = 0.001), and viral titers of cell culture supernatants were determined at the indicated time points. Bars indicate means ± SD obtained from three independent experiments performed in triplicates. *, P ≤ 0.05; **, P ≤ 0.01 by unpaired t test. (Right) The remaining fraction of the cells was lysed and tested by Western blotting for adequate knockdown and expression of viral proteins as shown. The position of a nonspecific band is highlighted by an asterisk. The lower part shows a quantification of protein expression from three independent experiments. Expression in the controls was set to 1. The means ± SEM are indicated. Download FIG S5, PDF file, 0.2 MB.Effect of the Striatin complex on replication of H1N1pdm09. MLE-15 cells were seeded and transfected 1 and 3 days later with siRNAs targeting mRNAs encoding Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The M1 protein functions as a multifunctional “Swiss army knife,” as it exerts different functions including the maintenance of virion integrity, inhibition of viral transcription, assistance in vRNP nuclear export, and endowment of virus assembly and budding , 42, 61.LKREI(T*)FHGAKE phosphorylation site is more likely to be phosphorylated by kinases belonging to the AGC kinase group, such as the PKC family members PDK1 and PKN1, which preferentially phosphorylate motifs containing a K/R at position −3 and an F at +1  were determined. Activity of the WT vRNP was set to 100%. Error bars represent SEM from three independent experiments. n.s., not significant. (Right) A portion of the cells was analyzed by immunoblotting for successful knockdown as shown. (B) Another fraction of the siRNA-treated cells was infected by SC35M (MOI = 3), and intracellular localization of the indicated proteins was determined by indirect immunofluorescence as shown. Nuclei were stained with Hoechst 33342 (blue). Pictures are representatives of two independent experiments. Download FIG S6, PDF file, 0.4 MB.Effect of STRN and STRN3 on polymerase activity and localization of viral proteins. (A) Expression of STRN and STRN3 was reduced upon knockdown with specific siRNAs in 293T cells. (Left) After knockdown, the cells were transfected to express a Pol I promoter-driven viral RNA-like transcript encoding the reporter gene together with pCAGGS plasmids, allowing the expression of mRNAs encoding the indicated viral proteins. One day later, cells were harvested and activities from firefly luciferase (generated by vRNP polymerase activity) and Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The antibodies, primers, and plasmids used in this study are provided in 10.1128/mbio.03231-22.4TABLE S4Table S4, DOCX file, 0.02 MB.Antibodies, primers, and plasmids used in this study. Download Copyright © 2023 Liu et al.2023Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2. Predesigned siRNAs targeting human or murine STRN and STRN3 were transfected into MLE-15 cells using Lipofectamine 3000 (Invitrogen) diluted in Opti-MEM (Life Technologies) according to the manufacturer's protocols. The medium was replaced with fresh growth medium 24 h.p.t. cells were then transfected for a second time at 48 h following the first transfection, using the same protocol. The cells were further incubated at 37°C for 48 h prior to either infection or lysis and Western blotting.Human embryonic kidney 293 T cells, Madin-Darby and canine kidney cells (MDCK-II), Vero-E6 cells, and murine MLE-15 lung epithelial cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown in an incubator at 37°C in a humidified atmosphere containing 5% (vol/vol) COThe WT SC35M virus and its M1 mutants were generated by using a set of eight plasmids based on the bidirectional pHW2000 plasmid reverse genetics system, described elsewhere . Plasmid2 upside down for 2 days. The plaques were harvested to infect MDCK-II cells for propagation of viruses from the purified plaques. Viral RNA was extracted from 140-μL aliquots of supernatants of virus-infected cells using a QIAamp viral minikit  according to the manufacturer’s protocol. The extracted viral RNA was reverse transcribed and amplified by PCR in a single reaction using a SuperScript IV One-Step reverse transcription-PCR (RT-PCR) system (Invitrogen). Briefly, 7.5 μL of extracted viral RNA was mixed with 25 μL 2× Platinum SuperFi RT-PCR master mix, 0.5 μL of enzyme mix, and M1-specific primers . The total volume was adjusted to 50 μL using nuclease-free water and subjected to cDNA synthesis at 55°C for 10 min, followed by predenaturation (98°C for 2 min) and PCR amplification. The different PCR products containing the SC35M M1 gene were purified from agarose gels and sequenced.Virus-containing culture supernatants from 293T cells transfected with nine plasmids coexpressing M1 WT and the M1 T108A mutant were analyzed by foci assays. Monolayers of MDCK-II cells in 6-well dishes were washed with phosphate-buffered saline (PBS) and infected with the virus-containing supernatant and various 10-fold serial dilutions in the infection medium (DMEM supplemented with 0.2% [wt/vol] BSA and penicillin-streptomycin) and incubated at 37°C for 1 h. The wells were aspirated to remove residual viral inoculum, and the MDCK-II cells were overlaid with 2 mL 1× agarose overlay mixture . After the agar overlay solidified, the plates were incubated at 37°C with 5% CO2, 0.9 mM CaCl2, 100 U/mL penicillin, and 100 μg/mL streptomycin; Life Technologies) at the indicated MOIs. Infections for multicycle propagation in MLE-15 cells were performed for 1 h at room temperature, and infections for single-cycle virus replication were performed for 1 h on ice. After the adsorption time, the inoculum was removed and monolayers were washed three times with PBS++. An appropriate amount of infection medium (DMEM supplemented with 0.2% [wt/vol] BSA and penicillin-streptomycin) was added, and cells were incubated for the indicated periods at 37°C. Infectious particles in the cell supernatants were quantified by foci assays on MDCK-II cells grown in 96-well plates.293T and MLE-15 cells were infected with IAV SC35M (H7N7) viruses or A/Hamburg/4/09 (H1N1) in PBS++/BSA  paraformaldehyde at room temperature for 8 min. After washing three times with PBS, cell membranes were permeabilized with 0.5% (vol/vol) Triton X-100, and unspecific antibody binding was blocked with blocking solution (PBS containing 10% [wt/vol] BSA and 0.5% [vol/vol] Triton X-100) at room temperature for 1 h. Cells were incubated with rabbit anti-M1, mouse anti-NP, mouse anti-M2, mouse anti-STRN, or mouse anti-STRN3 primary antibodies in PBS containing 1% (wt/vol) BSA and 0.5% (vol/vol) Triton X-100 at 4°C for 16 h. Cells were washed three times with PBS and incubated with Alexa Fluor 488- and Cy3-conjugated secondary antibodies for 1 h at room temperature. After incubation, cells were washed three times, and DNA was stained with 1 μg/mL Hoechst 33342. Coverslips were washed again and mounted on microscope slides with Mowiol mounting medium (Carl Roth). Slides were protected from light and stored at 4°C.293T cells were grown on coverslips in 24-well plates coated with poly-Confocal images were acquired with a Zeiss confocal LSM 710 microscope equipped with a Plan-Apochromat 63×/1.40 oil differential interference contrast M27 objective. Samples were illuminated using a diode (405-nm) laser, a DPSS (561-nm) laser, or an argon (488-nm) laser. Pictures were acquired and scale bars were calculated using the Zen Lite system . STRN3 superclusters were quantified for each image with prezoom of 0.6 at a frame size of 512 by 512 pixels, resulting in a resolution of 0.44 μm/pixel, and further analyzed in ImageJ. Threshold was set to 2× background intensity, and the “Analyze Particles” settings were size 0 to infinity, circularity 0.20 to 0.50. Fluorescence intensities were quantified with ImageJ and statistically analyzed using GraphPad Prism 9.g. The cells were resuspended in an appropriate amount of IGEPAL lysis buffer  and incubated on ice for 20 min. The lysates were cleared by centrifugation for 10 min at 4°C and 16,000 × g. The supernatant was then used for IP experiments or mixed with 5× SDS sample buffer, boiled at 95°C for 5 min, and analyzed by Western blotting as described elsewhere . Finally, peptides were dissolved in 25 μL of water with 5% (vol/vol) acetonitrile and 0.1% (vol/vol) formic acid. The mass spectrometric analysis of the samples was performed using a timsTOF Pro mass spectrometer . A nanoElute high-performance liquid chromatography system , equipped with an Aurora column (25-cm by 75-μm) C18 RP column filled with 1.7-μm beads (IonOpticks) was connected online to the mass spectrometer. A portion of approximately 200 ng of peptides corresponding to a 2-μL sample volume was injected directly on the separation column. Sample loading was performed at a constant pressure of 8 × 103 Pa. Separation of the tryptic peptides was achieved at a 50°C column temperature with the following gradient of water–0.1% (vol/vol) formic acid (solvent A) and acetonitrile–0.1% (vol/vol) formic acid (solvent B) at a flow rate of 400 nL/min: linear increase from 2% (vol/vol) B to 17% (vol/vol) B within 18 min, followed by a linear gradient to 25% (vol/vol) B within 8 min and linear increase to 37% (vol/vol) solvent B in an additional 3 min. Data analysis was performed using MaxQuant (version 1.6.17.0) with the Andromeda search engine, and UniProt databases were used for annotating and assigning protein identifiers (2 transformed. For the t test results and volcano plots in Samples bound to (magnetic) beads were washed three times with 100 μL 0.1 M ammonium bicarbonate solution. Proteins were digested “on-bead” by the addition of sequencing-grade modified trypsin (Serva) and incubated at 37°C for 45 min. Subsequently, the supernatant was transferred to fresh tubes and incubated at 37°C overnight. Peptides were desalted and concentrated using Chromabond Cntifiers . IntensiThe 293T cells were transfected using the 8-plasmid reverse genetic system expressing SC35M M1 WT or M1 T108A/E mutants and incubated for 24 h. For the knockdown experiments, 293T cells were transfected twice with siRNAs 48 h apart. Two days after the second transfection, cells were further infected with SC35M virus (MOI = 3) on ice and incubated at 37°C for 6 h. Cells were washed three times in cold PBS and subsequently cross-linked by the addition of DSS or DSG at a final concentration of 2 mM. The reaction mixture was incubated for 30 min at room temperature. After removal of DSS or DSG, the cross-linking was quenched by the addition of 900 μL of PBS containing 20 mM Tris at room temperature for 15 min. Cells were then washed twice in PBS and lysed in IGEPAL buffer prior to SDS-PAGE.Renilla luciferase activities were detected using the dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol. Renilla activities were used for data normalization.293T cells were transfected twice with siRNAs 48 h apart. Two days after the second transfection, cells were further transfected with pCAGGs plasmids encoding SC35M PB2, PB1, or PA (each 125 ng) or NP (800 ng) together with the firefly luciferase-encoding viral minigenome construct pHW72-Luci (25 ng) and a plasmid . At 24 h.p.t., the cells were harvested, washed, lysed for 15 min in 150 μL of 1× passive lysis buffer. Firefly and t test for two-group comparisons using the SPSS 19 program; P values of ≤0.05 were considered significant. Unless otherwise noted, error bars indicate the means and standard deviations of at least three independent experiments. Processing of confocal microscopy pictures and calculation of scale bars were done using Zen Lite software . Protein structures were analyzed using PyMOL (version 2.4.1.), and the STRING database (version 11.5) was used to reveal known and predicted interactions between the M1 interactors.Image Lab 6.0.1 (Bio-Rad) and ImageJ software were used for image acquisition and densitometric analysis of Western blot data. GraphPad Prism 9  was used to perform statistical analysis and generation of graphs. The statistical significance was calculated by using the unpaired Student's Viral infections were performed in accordance with German biosafety regulations pertaining to the propagation of IAVs, using biosafety level 3 containment laboratories approved for such use by the local authorities .


These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit molecular dynamics models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG) The standard collagen triple helix structure consists of three left-handed polyproline II helical chains twisted around each other to form a right-handed superhelix. This triple-helix structure is stabilized by the regular repeating sequence (Xaa-Yaa-Gly)perhelix , 3. The perhelix , 5, 6. n sequence has been found to be the cause of collagen-related diseases, including osteogenesis imperfecta, Ehlers Danlos syndrome IV, and Alports Disease  Disease . The rep Disease . In contproteins , 11.5-Gln-Gly-(Xaa-Yaa-Gly)10. In this triple-helix domain, the (Xaa-Yaa-Gly)n pattern is interrupted at one site by an incomplete triplet, QG, and similar breaks in repeating pattern have been shown to be highly destabilizing. Further, the triple-helices N terminal to the QG break self-associate to form MBL trimers to hexamers. MBL circulates in blood in complexes with the MBL-associated serine proteases (MASPs) , and these proteases bind to MBL through its collagen region, close to the Gly-Gln-Gly (or GQG) interruption , 135-Glnrruption , 15, 16.n units will form a stable triple-helix. To better define the molecular flexibility of the collagen triple-helix, an atomistic modeling approach, in combination with analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS), was applied to a set of peptides consisting of repeating POG triplets of different lengths. The results indicated a small degree of inherent nonlinearity for the longer (POG)n peptides, despite the close packing of the three chains and the rigid imino acid side chains  experiments combined with molecular dynamics (MD) modeling  to triplpeptides  providespeptides , varyingpeptides , 21, 22.yproline ; the G->utations ; and T3-utations  8, (POG)10, and (POG)12 were previously reported 13, and (POG)14 were again included as controls for the MBL peptide measurements.To study conformational flexibility in the MBL collagen stalks, four MBL-related peptides were designed that included the GQG interruption flanked either by POG tripeptides or by the native human MBL triplets . MBL-C . To invereported , and her10 has Pro in all Yaa positions, rather than the more stabilizing Hyp (PPG)10  (PPG)10 . The G >tability . Finallytability  , and a minimum at 197 to 198 nm, and the ratio of the ellipticity of the positive peak to the negative peak was close to that expected for a fully triple-helical peptide (A–C).Circular dichroism (CD) spectroscopy was carried out on the four MBL collagen peptides, as well as on a (POG)tability . Thus tem inset, A, and a  peptide . The int peptide , A–C.Figm obtained under the standard conditions in our lab reflect well with the comparative thermal stability of different triple-helical peptides. Both the MBL-N and MBL-C peptides formed triple helices and have melting temperatures of 21 °C and 32 °C, respectively (A). In contrast, the control peptide (POG)10 showed a higher melting temperature of 64 °C, which compares well with an earlier value of 60 °C (A). To check the effect of the Pro or Hyp residue next to the GQG interruption, the MBL-N peptide was compared with a slightly modified MBL-E peptide in order to compare the same number of POG repeats in both peptides (B). The replacement of Pro with Hyp increased the melting temperature from 21 °C to 30 °C, showing a stabilization caused by Hyp in MBL-E. Finally, MBL-C and MBL-E were compared to see whether the positioning of the POG triplets before or after the GQG interruption conferred stability (C). There, both peptides showed almost the same melting temperature of 29 °C and 32 °C respectively, thus the positioning of the POG triplets had little effect on stability. For completion, the corresponding Tm values of 33 °C, 29 °C, and 20 °C from our earlier studies of (PPG)10, G > A and T3-785, respectively, were included in The melting curves were obtained under identical conditions, at a very slow heating rate, as previously described . Extensiectively . The MBLectively A. In conof 60 °C  and showof 60 °C A. To chepeptides B. The retability C. There,10 was previously measured as 70 °C  was performed on the MBL peptides to confirm the outcome of the CD studies . Single as 70 °C , 30, whias 45 °C . The thenditions . There wFirst, experimental data on the 10 collagen peptides in solution was acquired by a combination of AUC, SAXS, and SANS. Second, 12,000 physically realistic atomistic models for each of these 10 collagen triple helices were produced using MD. A comparison through filtering of these theoretical models against the experimental SAXS and SANS data identified the best-fit structures for each collagen triple helix in solution. This procedure followed our procedures for atomistic scattering modeling , 20.A and C). All 10 peptides formed stable triple helices in both phosphate and histidine buffers at 20 °C. The size distribution c(s) analyses resulted in good fits to the sedimentation boundaries and produced single narrow distinct peaks for each collagen helix demonstrating structurally monodispersity (B and D). The root-mean-square residuals from SEDFIT were satisfactory at 0.01 to 0.02 for the interference data fits and up to 0.04 for the absorbance data fits. In all 10 cases, the absence of peaks at the lowest S values confirmed that there was no dissociation of the helices into single peptides. The experimental sedimentation coefficient s20,w values were calculated from the peak positions in the c(s) analyses. For the MBL peptides, their averaged s20,w values were 1.20 S, 1.15 S, 1.09 S and 1.30 S for MBL-C, MBL-N, MBL-E, and MBL-12, respectively, in 137 mM PBS buffer 10, (POG)13, (POG)14, and (PPG)10, respectively, in 137 mM PBS  repeats, which reflected the combination of both the increasing mass and lengths of the helices. The proline-rich peptide (PPG)10 sedimented slower (∼0.33 S less) than the equivalent length collagen peptide (POG)10. The average rates of sedimentation for the G > A and T3-785 peptide were 1.19 S and 0.95 S, respectively. However, (PPG)10 sedimented almost at the same rate as the Type III T3-785 peptide of the same length, suggesting similar hydrodynamic properties for these two peptides 10, and (POG)13, respectively   compared to the theoretical values for linear structures (unfilled data points) is consistent with some bent nonlinear collagen structures existing in solution  . The AUCsolution .Figure 5Rg, a measure of macromolecular elongation, were derived . For (POG)14, the initial Guinier plots indicated high aggregation from the increased intensities at low Q values, and this was excluded from further analysis. For the two (POG)n peptides, the Q ranges of the Guinier fits were successively reduced from 0.15 to 0.57 nm−1 for (POG)10 to 0.21 to 0.45 nm−1 for (POG)13. For the remaining three peptides, the Q ranges of the Guinier fits were all similar between 0.16 and 0.56 nm−1 for (PPG)10, 0.18 and 0.56 nm−1 for G > A, and 0.20 and 0.57 nm−1 for T3-785. Overall, the Guinier regions became reduced with increased length of the collagen peptides. The SAXS experimental Rg values calculated from the Guinier analyses ranged between 1.8 nm and 2.8 nm 10 here with that reported previously 13. The Guinier plots for (POG)14 indicated excessive aggregation again at low Q values and were also excluded from further analysis. For neutrons, the Q ranges of the individual Guinier fits were similar (B). The SANS experimental Rg values were 1.97 nm for MBL-C, 2.02 nm for MBL-N, 2.13 nm for MBL-E, 2.81 nm for MBL-12, 2.22 nm for (POG)10, and 2.80 nm for (POG)13, again with low standard deviations  similar B. The SAviations . The tree length  supporteP(r) of the full-length scattering I(Q) curves provided real space determinations of the Rg values (P(r) curves was affected by noise in the I(Q) datasets that was attributed to the comparatively low molecular masses in use for the scattering experiments (P(r) curves were obtained with Rg values that were comparable with the Guinier Rg values. The P(r) analyses provided the maximum length L of each collagen helix which corresponded to the value of r when the P(r) curve reaches zero. From the SAXS analyses, the maximum length L values were 8.7 nm for MBL-C, 7.1 nm for MBL-N, 10 nm for MBL-E, 12.5 nm for MBL-12, 8.5 nm for (POG)10, 9.5 nm for (POG)13, 9 nm for (PPG)10, 11 nm for G > A, and 10 nm for T3-785, respectively (P(r) peak maximum provided values for M, the most common interatomic distance in each collagen molecule, and its value reflects the width of the collagen triple helix. Here, the M values were 0.86 nm for MBL-C, 0.81 nm for MBL-N, 0.81 nm for MBL-E, 0.94 nm for MBL-12, 0.88 nm for (POG)10, 0.90 nm for (POG)13, 1.28 nm for (PPG)10, 0.86 nm for G > A, and 0.90 nm for T3-785 respectively. For MBL-12, the relatively small L and larger M values suggested some bending with increase in length. The increases in the L values were consistent with the increased length of the collagen peptides.Pair-distance distribution analyses g values . The apperiments . Nonetheectively . The P(rP(r) analyses, the L values were 7.5 nm for MBL-C, 7.6 nm for MBL-N, 9.7 nm for MBL-E, 12.5 nm for MBL-12, 10.5 nm for (POG)10, and 10.0 nm for (POG)13 (P(r) curves gave Rg values that were comparable with the Guinier Rg values, although the comparison was against limited by noise in the I(Q) curves. The trends in the SANS L values followed that of the SAXS L values. Minor perturbations resulted in the form of an abrupt end to the P(r) curve at large r values or small extra P(r) peaks (B). The M values were 0.47 nm for MBL-C, 0.45 nm for MBL-N, 0.49 nm for MBL-E, 0.53 nm for MBL-12, and 0.57 nm for (POG)10. The SANS M values were noticeably reduced in heavy water compared to the SAXS data, and this was attributed to the near invisibility of the hydration shell in SANS experiments with heavy water (From the corresponding SANS  (POG)13 . The P(rr) peaks B. The M vy water .Rg calculation from the scattering curve I(Q) at low Q values. The experimental SAXS and SANS Rg values were compared with the theoretical Rg values calculated from the crystal-derived linear models 10, 2.90 nm for (POG)13, 2.32 nm for (PPG)10, 2.42 nm for G > A, and 2.27 nm for T3-785  curves were superimposed upon each other. The goodness-of-fit R factor monitors the degree of deviation between the two scattering curves. Visually the superimposition showed good agreements (A and B). However, on comparing the SAXS curves against those for the linear structures, the R factors were comparatively larger than expected. They were 5.0% for MBL-C, 14.3% for MBL-N, 12.0% for MBL-E, 7.4% for MBL-12, 4.2% for (POG)10, 7.9% for (POG)13, 5.7% for (PPG)10, 3.3% for G > A, and 9.1% for T3-785 10, and 13.4% for (POG)13. Given that the crystal-derived structures were very close to linear, the higher R factors and poorer curve fits with increasing peptide length suggested nonlinearity in the helices  curve with the theoretical curves from the models in each simulation enabled the identification of the best-fit solution structural models for each peptide. A 50 ns MD simulation at 300 K produced 12,000 models with stereochemically correct conformations for each of the nine collagen peptides  curve for each of the 12,000 collagen models was computed using experimental curve parameters using SCT software (I(Q) curves with the experimental I(Q) curve through the goodness-of-fit R factors (R factors represented the better fit structures. The independent SAXS and SANS fits are each considered in turn:(i)R factors whose Rg values at the minima were within range of the experimental SAXS Rg values . That for MBL-N gave higher R factors for reason of the noisy experimental SAXS data (A). The MD structures gave notably lower R factors (blue) than the linear model (red) for the four MBL peptides, showing that better curve fits were obtained from many MD structures compared to the linear starting models 13, (PPG)10, G > A, and T3-785 peptides, the R factors for four of the five of the best-fitting MD structures were slightly lower than those for their linear starting models, indicating that the MD fits had mostly given improved structures compared to the linear starting models.The nine SAXS fits each resulted in a clustered distribution of AXS data A. The MDg models . For the(ii)Rg values (B). Three of the four MD best-fit MBL structures gave R-factors (blue) that were less than those for the four linear starting models (red) (10 and (POG)13 fits were likewise improved by the use of the MD curve fits.The six SANS fits again resulted in clustered distributions whose minima were within range of the experimental SANS g values B. Three ls (red) . The  curves from the best-fit MD-modeled structures were now compared against their experimental I(Q) curves (P(r) curve comparisons of the experimental data and the linear and MD models  curves . VisuallD models . Generalo seen in both the SAXS and SANS best-fit structure for MBL-N which corresponded to the native MBL sequence with eight residues EPGQGLRG flanked by POG repeats. Smaller bends of 19°, 18°, and 11° were seen at the GQG interruptions at the centers of the other three MBL peptides MBL-C, MBL-E, and MBL-12. In contrast, the four MBL models derived from crystal structures showed smaller bends of 4°-9°. For the (POG)10, (POG)13, (PPG)10, G > A, and T3-785 structures, there were reduced differences between the linear starting models (bends of 1° to 3°) and the experimental best-fit structures of 6o-11o, apart from an increased bending of 22° seen for the best-fit (PPG)10 structure n peptides peptides , 20, 38.ormation .10 and (POG)13 were comparatively linear with bends of 6° and 11° and show the role of the imino acids in structural stability. The synthetic collagen peptide (PPG)10 with no hydroxyproline residues displays reduced thermal stability (melting temperature Tm 24.5 °C) compared to (POG)10 (Tm 60 °C). The thermal stability of (PPG)10 is thus dramatically lower than (POG)10, consistent with the stabilizing stereoelectronic and enthalpic effects of Hyp (10 compared to (POG)10 and (POG)13. Interestingly, the high resolution crystal structure of the (PPG)10 triple helix is indistinguishable from that for (POG)10, both having a bend of 2° (10 and (POG)10 peptides differ significantly in their linearity s of Hyp , and thend of 2° , 28, 29,inearity .10 context, and this break in the repeating tripeptide pattern results in a major loss of triple-helix stability 13, and (POG)14 peptides resulted in slightly bent structures under near physiological conditions in contrast with the starting linear crystal structures. These structures showed similar bending compared to our previous solution structures for (POG)6, (POG)8, (POG)10, and (POG)12  (POG)12 , 42.Functionally, sequence variability in the collagens can affect recognition and/or conformation in disease-causing mutations of the complement proteins. The MD approach  offers a10, (POG)13, (POG)14, (PPG)10, G > A, and T3-785), which were unblocked or blocked at both termini (acetylated N terminus and amidated C terminus), were synthesized using FastMoc Chemistry at the Tufts University Core Facility. Peptides were purified by HPLC, and their identity was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Peptide concentrations were determined by an ultraviolet-visible spectrophotometer  with an extinction coefficient εTyrosine 280 of 1280 M−1 cm−1. Other peptides were purchased from Pepceuticals Ltd and Peptide Institute Inc. All peptides were obtained as a dry powder and used without any further purification (214 = 2200 cm−1 M−1 per peptide bond.Ten highly purified (>98%), monodisperse collagen peptides fication . When rem) is defined as the temperature at which the fraction-folded F(Tm) is equal to 0.5 as described previously . Wavelength scans were collected from 190 to 260 nm at 0 °C. Temperature scans were monitored by measuring the molar residue ellipticity at 220 nm from 0 to 70 °C with a 10-s averaging time and 1.5-nm bandwidth. Samples were equilibrated for 2 min at each temperature, and the temperature was increased at an average rate of 0.1 °C/min. The melting temperature . Each sample was dialyzed against PBS overnight before measurement. Dialysis buffer was collected and used as reference for the corresponding sample. Samples were loaded into the cells at 0 °C and heated at a rate of 1 °C/mm till 70 °C.Sedimentation velocity AUC experiments were performed using two Beckman XL-I analytical ultracentrifuges (Beckman Coulter Inc) equipped with an eight-hole AnTi50 rotor with standard double-sector cells with column heights of 12 mm. Prior to experiments, the peptides were allowed to assemble into triple helices by allowing them to equilibrate in solution for at least 48 h at room temperature. The samples were placed in velocity cells with two-sector aluminum centerpieces. Sedimentation was monitored at a rotor speed of 50,000 rpm for ∼14 h using absorbance and interference optics at 222 nm.2O  in H2O, 20 mM L-histidine  in H2O, or in 20 mM L-histidine buffer with 2H2O. The samples were dialyzed using dialysis cassettes  with three buffer changes. With H2O, the concentrations of 10 peptides used in AUC experiments ranged between 0.25 and 1 mg/ml for MBL-C, 0.25 and 1 mg/ml for MBL-N, 0.25 and 1.25 mg/ml for MBL-E, 0.25 and 1.25 mg/ml for MBL-12, 0.25 and 1 mg/ml for (POG)10, 0.20 and 0.75 mg/ml for (POG)13, 0.40 and 0.80 mg/ml for (POG)14, 1 and 2.5 mg/ml for (PPG)10, 0.40 and 0.95 mg/ml for G > A, and 0.3 and 1.1 mg/ml for T3-785, respectively. The concentrations of six peptides used in the AUC experiments in 2H2O ranged between 0.94 and 3.7 mg/ml for MBL-C, 1.5 and 4 mg/ml for MBL-N, 0.75 and 4 mg/ml for MBL-E, 1 and 4 mg/ml for MBL-12, 1 and 4 mg/ml (POG)10, and 1 and 3.5 mg/ml for (POG)13. Buffer densities were measured using an Anton Paar DMA 5000 density meter. At 20 °C, the densities were 1.00543 g/ml and 1.00578 g/mol for the 137 mM PBS and 20 mM L-histidine buffers, respectively. The viscosities of the buffers were measured using an Anton Paar AMVn automated microviscometer. The viscosities at 20 °C were 0.01683 P and 0.01019 P for 137 mM PBS and 20 mM L-histidine buffers, respectively. The density and viscosity for 20 mM L-histidine buffer in 2H2O at 20 °C were 1.11100 g/ml and 0.01384 P, respectively.The AUC data were collected at 20 °C in either 137 mM PBS prepared in deionized Hc(s) analyses were performed using SEDFIT software version 15.10 b (c(s) analyses which provided the sedimentation coefficients s20,w and mass values. The c(s) analyses fitted the sedimentation data using the Lamm equation, which defines the transit of a solute under a gravitational force in a sector shaped cell. Prior to analysis, the partial specific volumes n and (PPG)10, 0.738 ml/g for G > A, and 0.741 ml/g for T3-785 collagen molecules. The c(s) algorithm assumes that all species have the same frictional ratio f/f0 within each fit. To optimize the fits, the values for f/f0, , the meniscus, the bottom of the cell and the base line were floated, until the overall root mean square deviation of the c(s) fits were below 0.01, and the visual appearance of the fits were reasonably satisfactory at low resolution (set at 50) and confidence (F) ratio (0.68). The final SEDFIT analyses used a fixed resolution of 200 and F ratio of 0.95. The observed s values were corrected to s20,w by:s is the sedimentation coefficient, the subscripts T,B refers to the temperature of the buffer. 20,w refers to water at 20 °C. ρ is the solvent density, η is the solvent viscosity, and The size distribution  15.10 b , 49 to dram SLUV . HoweverQ range from 0.04 nm-1 to 4 nm-1 . Potential radiation damage was monitored during data acquisition to confirm its absence. The final scattering data were collected for collagen peptides ranging from 0.25 to 8 mg/ml in 20 mM L-histidine buffer at 20 °C. After in-house data reduction, buffer measurements were averaged and subtracted, from each of the sample frame using a Java-based program SCÅTTER (version 3.0) (http://www.bioisis.net)  . The resQ range of ∼0.05 to 10.0 nm−1. The beam operated in time-of-flight mode with a beam diameter of 12 mm and an incident wavelength range of 0.175 to 1.65 nm. 800 μl of collagen peptide samples, each prepared at concentration between 4.0 and 5.5 mg/ml in 100% deuterated 20 mM L-histidine buffer, were measured in 2 mm-thick disc-shaped Hellma quartz banjo cells positioned in a temperature-controlled rack at 20 °C. This condition showed the collagen peptides in a high negative solute-solvent contrast  of the pulsed neutron source ISIS at the Rutherford Appleton Laboratory . A pulsecontrast . Data cocontrast . The MANch pulse .Rg, the molecular mass and structural dimensions. In a given solute–solvent contrast, the radius of gyration Rg corresponds to the average distance of each scattering elements from their center of scattering and is a measure of structural elongation. Q is the angular difference between the incident and scattered beams. Guinier analyses at low Q values  give the Rg value and the forward scattering at zero angle I(0) from the expression which is valid in a Q.Rg range up to 1.3 =IRg values were determined using the SCT suite of open source software tools  is acquired in inverse space, the structure cannot be interpreted from visual inspection alone. Fourier transformation of the scattering intensity curve I(Q) in reciprocal space into real space gives the distance distribution function, P(r). The P(r) curve corresponds to the distribution of distances r between the elements. This yields the maximum dimension of the macromolecule L and its most common distance vector M in real space.The re tools  and wereP(r) curve (GNOM software (version 4.6) was used to calculate the r) curve .3B0S), which has a 7/2 helical conformation and a (GPO)9 repeat unit 9. Modeling was performed using PyMol software . The MBL-C linear model was generated using (POG)10 as the starting linear model by removing a Pro residue in the fifth triplet and substituting Hyp with Gln in the fifth triplet residues in all three chains. The MBL-N linear model was created using MBL-C as the starting model by replacing the POG residues in the fifth and seventh triplets to EPG and LRG, respectively. The MBL-E linear model was created using MBL-N as the starting model by adding POG at the N terminus and substituting Pro with Hyp in the sixth triplet of all three chains. The MBL-12 linear model was created by the addition of (POG)2 at the C terminal of MBL-E and substituting Hyp with Pro residue in the sixth triplet of all three chains. The linear models for (POG)13 and (POG)14 were created using (POG)10 as the starting linear model by adding three and four POG triplets, respectively. Crystal structures for (PPG)10 (PDB ID: 1K6F), G > A (PDB ID: 1CAG), and T3-785 (PDB ID: 1BKV) were available (2) groups were added to all the above collagen helices to create the blocked ends except for (PPG)10.Starting models for the collagen triple helices were constructed from a linear crystal structure of a collagen-like peptide 13, (PPG)10, G > A, and T3-785) were generated using the THeBuScr, version 1.07 software package  did not qualitatively change the results of the Rg analyses. All MD simulations were carried out using the GROMACS v4.5.5 software package with the AMBER99sb-ILDNP force-field  package . The N ace-field . Other sce-field . The bence-field  were comhttp://sassie-web.chem.utk.edu/sassie2) and SCT software n peptides meant that additional experiments using SAXS were needed to confirm these solution structures.Theoretical sedimentation coefficients HYDROPRO . HYDROPRHYDROPRO , using avergence . The exp± 0.21 S , the lowhttps://www.sasbdb.org/) with the reference codes MBL-C, SASDRS2; MBL-N, SASDRT2; MBL-E, SASDRU2; MBL-12, SASDRV2; (POG)10, SASDRW2; (POG)13, SASDRX2; (PPG)10, SASDRY2; G>A, SASDRZ2; T3-785, SASDR23.All data are contained within this manuscript. The 10 best-fit models for the nine peptides corresponding to the X-ray fit searches at 1.00 mg/ml  are avaiThis article contains The authors declare that they have no conflicts of interest with the contents of this article.


SVK) constant reflects the binding affinity, indicating a moderate interaction (104 M−1) being spontaneous (ΔG°= -25.0 kJ/mol at 296 K), enthalpically (ΔH° = -9.31 ± 1.34 kJ/mol), and entropically (ΔS° = 52.9 ± 4.4 J/molK) driven. Binding causes only a very weak perturbation on the secondary structure of albumin. There is just one main binding site in HSA for 4-TPyP (n ≈ 1.0), probably into the subdomain IIA (site I), where the Trp-214 residue can be found. The microenvironment around this fluorophore seems not to be perturbed even with 4-TPyP interacting via hydrogen bonding and van der Waals forces with the amino acid residues in the subdomain IIA.The interaction between human serum albumin (HSA) and the non-charged synthetic photosensitizer 5,10,15,20-tetra(pyridine-4-yl)porphyrin (4-TPyP) was evaluated by in vitro assays under physiological conditions using spectroscopic techniques  combined with in silico calculations by molecular docking. The UV-vis and steady-state fluorescence parameters indicated a ground-state association between HSA and 4-TPyP and the absence of any dynamic fluorescence quenching was confirmed by the same average fluorescence lifetime for HSA without (4.76 ± 0.11 ns) and with 4-TPyP (4.79 ± 0.14 ns). Therefore, the Stern–Volmer quenching ( Human Serum Albumin (HSA) is the most abundant globular protein in the bloodstream 35–50 g/L). It is synthesized in the liver and is responsible for the transport of both endogenous and exogenous compounds, e.g., fatty acids, hormones, metabolites, and commercial drugs to their target ,2. For t5–50 g/L.∆ = 0.76, in acetonitrile) and phototoxicity in a nanomolar scale under green light irradiation (522 nm) with very low light dose (1.0 J/cm2) -pyridinium-4-yl)porphyrin (4-RuTPyP) , SVK, and qk are the porphyrin concentration, Stern–Volmer quenching constant, and bimolecular quenching rate constant, respectively. The τ0 is the obtained experimental average fluorescence lifetime for HSA without 4-TPyP in PBS (τ0 = 4.76 ± 0.11 ns), while aK and n are the binding constant and number of binding sites, respectively. The ΔH°, ΔS°, and ΔG° are the enthalpy, entropy, and Gibbs’ free energy change, respectively. Finally, T and R are the temperature  and gas constant (8.3145 Jmol−1K−1), respectively.To obtain quantitative parameters on the binding capacity of HSA:4-TPyP, the maximum fluorescence data after inner filter correction were treated by Stern–Volmer (Equation (2)), double-logarithmic (Equation (3)), van’t Hoff (Equation (4)), and Gibbs’ free energy (Equation (5)) approximations ,45,46,47−5 M, in PBS) without and with 4-TPyP (1.32 × 10−5 M) at room temperature. The instrumental response function (IRF) was collected using a Ludox® dispersion.Time-resolved fluorescence decays were obtained for 3.0 mL of HSA solution  without and upon successive additions of 4-TPyP in the same concentrations of porphyrin used in the steady-state fluorescence studies  at room temperature in the range of 260-320 nm for Tyr (Δλ = 15 nm) and 240-320 nm for Trp (Δλ = 60 nm). Finally, the 3D-fluorescence spectra were recorded at the 240–320 nm range for 3.0 mL of HSA solution  without and upon addition of 4-TPyP (1.32 × 10−6 M) using λexc = 200–360 nm and λem = 200–460 nm, at room temperature.The SF spectra were obtained for 3.0 mL of has solution  were normalized and expressed as the mean residue weight ([Θ]MRW) in deg. cm2-dmol−1, defined as [Θ]MRW = (Θobs × 10−3) × 100 × MW/(l × c × NAA), where MW is the protein molecular weight, c is the protein concentration in milligrams per milliliter, l is the light path length in centimeters, and NAA is the number of amino acids per protein. The secondary structure content was estimated by analysis of the CD spectra using the online server BestSel (Beta Structure Selection http://bestsel.elte.hu/index.php (accessed on 2 June 2022)).The CD spectra (200–260 nm) were recorded for HSA without and with 4-TPyP in PBS at 310 K. The HSA concentration was fixed at 1.0 × 10The 4-TPyP structure was built and energy-minimized by the Density Functional Theory (DFT) method with the potential B3LYP and basis set 6-31G*, available in the Spartan’14 software . The crystallographic structure of HSA was obtained in the Protein Data Bank (PDB), with access code 3JRY . MoleculChemPLP’, which is the default function of GOLD 2020.2 software. Figures for the docking poses were generated using PyMOL Delano Scientific LLC software .Hydrogen atoms were added to HSA according to the data inferred by GOLD 2020.2 software on the ionization and tautomeric states at pH 7.4. Docking interaction cavity in the protein was established with 8 Å radius from the amino acid residues Trp-214, Tyr-411, and Tyr-161 for sites I, II, and III, respectively. The number of genetic operations  in each docking run was set to 100,000. The scoring function used was ‘


Plasma level of polysaccharide (1 → 3)-β-D-Glucan (βDG), as a diagnostic marker of invasive fungal infection has been reported to be elevated in people living with HIV (PLWH). We assessed the association of circulating βDG to inflammation and systemic immune activation and the effect of antiretroviral therapy (ART) on βDG in PLWH.Plasma and peripheral blood monocular cell samples from 120 PLWH naive to ART and after 1 year's ART were collected. Plasma levels of βDG, markers of bacterial translocation, gut damage, and cellular immune activation were quantified.r = −0.25, p = 0.005) and positively with HIV viral load  before ART. It was also positively correlated with immune activation markers, including PD-1 expression on CD4+ T cell  and CD8+ T cell , as well as HLADR+CD38+ co-expression on CD8+ T cell , but not with the plasma levels of LPS , LPS binding protein , soluble LPS receptor sCD14 , intestinal fatty acid binding protein , and regenerating islet-derived protein 3α . After 1 year's ART, the levels of βDG were significantly decreased compared to that in pre-ART .The plasma βDG levels were negatively correlated with CD4+ T cells count (The level of plasma βDG was associated with cellular immune activation and decreased after ART in PLWH, suggesting it could serve as a biomarker of immune activation and efficacy monitoring. Increased T-cell turnover, elevated serum levels of pro-inflammatory cytokines and chemokines, and altered gut microbiome translocation were major characteristics of HIV infection –3. MicroFungal cell walls contain polysaccharides that are absent in humans. As one of the major components of fungal cell walls, (1 → 3)-β-D-Glucan (βDG), is a useful target for assessing invasive fungal in circulation . CurrentThe levels of immune activation aim at identifying associations between the relevant biomarkers and clinical outcomes. In our report, we accessed the association of βDG with other immune activation markers, bacterial translocation, and gut damage. We further quantified the dynamic changes of levels of βDG in PWLH initiating ART.Blood samples from participants in a prospective, randomized, clinical trial were collected . All theSequential blood samples from a total of 120 participants were analyzed. Plasma βDG was measured by the Fungitell Limulus Amebocyte Lysate (LAL) assay  according to the manufacturer's instruction. Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify plasma LPS , LPS binding protein , soluble LPS receptor CD14 (sCD14), intestinal fatty acid binding protein (IFABP), regenerating islet-derived protein 3α (REG3α), and soluble CD163 .To detect the correlation between βDG levels and immune activation before ART, we investigated T-cell activation by measuring PD-1 expression on CD4+ and CD8+ T cells –15 and aSD) and compared using t-tests; continuous data with skewed distribution were expressed as median  and compared using the Kruskal–Wallis test. Categorical data were expressed as frequencies and percentages and compared using the chi-square (χ2) test. The correlation analysis was performed using the non-parametric Spearman test. The p < 0.05 was statistically significant.Data were analyzed using IBM SPSS version 23 and GraphPad Prism 8.0 software. Continuous data with normal distribution were expressed as means ± standard deviation  age of participants was 28.5 (25–35) years and 92.5% were male. The median Pre-ART HIV RNA was 4.47 (4.02–4.81) Log10 copies/ml. The median CD4 T-cell count was 287.00 (193.00–411.00) cells/μl, and it was improved after ART to 470.50 (338.00–657.75). All the participants reached viral suppression after 1 year's ART. All the characteristics of the 120 participants were described in r = −0.25, p = 0.005; r = −0.34, p = 0.0002; r = 0.28, p = 0.002; r = 0.40, p = 0.01; r = 0.47, p = 0.002; r = 0.56, p = 0.0002; In PLWH naive to ART, the βDG levels were negatively correlated with CD4 T cell count , LBP , sCD14 , nor did associated with markers of gut damage, such as IFABP  and REG3α   .p < 0.001; r = −0.25, p = 0.005); participants were then further classified into two subgroups depending on the CD4+ T cell count (300 cells/μl). We evaluated the correlations between βDG levels and ART in CD4 > 300 cells/μl group and CD4 ≤ 300 cells/μl group, respectively. The βDG levels decreased notably after ART in CD4 > 300 cells/μl group  vs. 18.80 (10.90–30.10), p < 0.0001, p < 0.0001, p = 0.07; 6.39 (6.00–6.66) Log10 pg/ml vs. 6.36 (6.09–6.65) Log10 pg/ml, p = 0.28; 4.14 (3.90–4.42) Log10 pg/ml vs. 4.17 (3.87–4.36) Log10 pg/ml, p = 0.28, For the bacterial translocation and gut damage markers, we only found that LBP decreased significantly after ART . The patients/participants provided their written informed consent to participate in this study.JC and HL were responsible for designing the study and revising the manuscript. JX, SG, and YX handled the specimens, did the experiments, analyzed the data, and wrote the manuscript. RC, RZ, YS, LL, and JW participated in the conduct of the study, including the recruitment, and follow-up of participants. RC was responsible for collecting the clinical data. QT directed and helped with the data analysis. XZ and DL assisted with ELISA experiments. All authors reviewed the article for intellectual content, contributed to the article, and approved the submitted version.This work was supported by Shanghai Shenkang Hospital Development Center (16CR1018A), the People's Republic of China (2017ZX09304027 and 2017ZX10202101), the Shanghai Science and Technology Committee (19YF1441300), and Shanghai Municipal Health Commission .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cydonia oblonga is a medicinal plant that is used to treat a number of health complications in traditional medication systems. The objective of this study was to evaluate the phytochemical composition, and antibacterial, antioxidant, and ant-diabetic potentials of methanolic extracts of Cydonia oblonga bark. The Cydonia oblonga bark extraction was fractionated through HPLC and seven purified fractions labeled as F1, F2, F3, F4, F5, F6, and F7 were obtained. The HPLC-UV analysis of methanolic extract showed the presence of a number of possible compounds. The GC-MS and HPLC analysis confirmed the presence of the following bioactive compounds in the crude extract and purified fractions: malic acid, mandelic acid, quercetin, caffeic acid, catechin hydrate, as morin , BIS-(2-ethylhexyl)phthalate and diisooctyl phthalate (F1), carbamide , octasiloxane and dimethylsiloxanecyclictrimer (F3), silicic acid and cyclotrisiloxane (F4), 6-AH-cAMP, 4H-cyclopropabenzazule, and 4-(4-chlorophenyl)-3-morpholinepyrol-2-yl)-butenedioic acid (F5), isopropyamine (F6), and 1-propylhydrazine (F7). The extract and purified fractions were then tested for biological activities. All the purified fractions and methanolic extract showed effective antibacterial activity; however, the highest activity was recorded for methanolic extract against Staphylococcus aureus and Streptococcus pneumonia. Antioxidant evaluation of methanolic extract and purified fractions against DPPH showed strong % inhibition of the synthetic free radical. The methanolic extract exhibited 87.41 ± 0.54% inhibition whereas fractions showed: F1, 85.45 ± 0.85; F2, 65.78 ± 0.68; F3, 58.61 ± 0.58; F4, 80.76 ± 0.59; F5, 571.29 ± 0.49; F6, 85.28 ± 0.94; and F7, 48.45 ± 0.62% inhibition. Ascorbic acid (standard) was used as a control with 94.88 ± 0.56% inhibition at a maximum concentration of 1000 µg/mL. The α-glucosidase inhibition assay of methanolic extract and purified fractions at a maximum concentration of 1000 µg/mL showed activities as: methanolic extract, 78.21 ± 0.67; F1, 55.01 ± 0.29; F2, 56.10 ± 0.24; F3, 62.44 ± 1.03; F4, 70.52 ± 0.15; F5, 62.18 ± 0.92; F6, 72.68 ± 0.2; and F7, 57.33 ± 0.05% inhibition. α-Amylase % inhibition of methanolic extract and purified fractions were noted as: methanolic extract, 77.98 ± 0.57; F1, 79.72 ± 0.02; F2, 79.72 ± 0.02; F3, 82.16 ± 0.48; F4, 77.37 ± 0.28; F5, 72.14 ± 0.30; F6, 74.24 ± 0.29; and F7, 56.58 ± 0.10 at the highest concentration of 1000 µg/mL. Acarbose (standard) showed 87.65 ± 0.71% inhibition of α-glucosidase and 85.99 ± 0.44% inhibition of α-amylase at the highest concentration of 1000 µg/mL. It was found that all biological activities of methanolic extract and purified fractions might be attributed to the fact that they are rich sources of phenolic and flavonoids along with other bioactive compounds. The total phenolic and flavonoid contents of methanolic extract were recorded higher as compared to purified fractions (TPC = 70% and TFC = 69%). Amongst the purified fractions, fraction 6 exhibited the highest TPC value (64%), and purified fraction 1 exhibited the highest value of TFC (58%). Recent research demonstrated that Cydonia oblonga may be considered an antibacterial medicinal plant. The result of the present study revealed that it might be utilized for the isolation of bioactive phytochemicals that can lead to new opportunities in the discovery of new antibiotics. Much work has been carried out on studying and isolating natural compounds with pharmacological, pesticidal, and dietary benefits. Natural products have attracted a lot of attention in research. Since the earliest days, natural products have been employed as therapeutic agents for curing diseases . The WHOSecondary metabolites are important components of the human diet as they are present in daily food products such as coffee, tea, soybean, fruits, and vegetables . Due to Antimicrobial resistance has caused serious concern for both human health and the control of bacterial infectious illnesses . Due to Cydonia oblonga) belongs to the family Rosaceae, which is conventionally popular for medicinal, nutritional, and ornamental uses [Quince  at the maximum concentration 1000 µg/mL showed 87.65 ± 0.71 α-glucosidase inhibition with 30 µg/mL IC50 value.The IC50 values of the purified fractions and Met + Ext were calculated at different concentrations along with percent inhibition values and are depicted in 50 value. Standard acarbose at the maximum concentration 1000 µg/mL showed 85.99 ± 0.44 α-glucosidase inhibition with 32 µg/mL IC50 values.The ICCydonia oblonga are a rich source of phenolic compounds, which confirms our observed results. Scientific interest in the standardization and characterization of herbal medications is ongoing in the herbal drug sector. Simple, rapid, practical, and affordable standardization approaches are becoming more and more desirable as sophisticated chromatographic techniques are introduced [Cydonia oblonga bark isolated fractions, and is one of the most extensively used techniques for separating phytoconstituents. About 25 phytochemical substances in the leaves and rhizome of Amomum nilgiricum species have been identified previously through GC-MS [The analysis of the present study indicated that Met-Ext had the highest TFC and TPC values of 69% and 70%, respectively. Purified fraction 6 had the highest TPC of 64% and purified fraction 1 had the highest TFC of 58%. According to Erdoğan et al. , the leatroduced . For thetroduced . The HPLtroduced . The GC-gh GC-MS . The GC-50 values of 600 and 800 µg/mL, respectively, whereas seed extracts were reported to have a lower antioxidant potential [Cydonia obolonga extract and the isolated fractions showed effectiveness against α-glucosidase and α-amylase. A previous study showed that plant extracts (methanolic and n-hexane) display remarkable anti-diabetic activity against α-glucosidase and α-amylase [Cydonia oblonga leaves in hydro-ethanolic extract was studied in normal and streptozotocin-induced diabetic rats. There was no significant effect on normal rat glucose, while a significant reduction in blood glucose levels was recorded in diabetic rats at the time period of 0 to 3 h [Antibiotics are widely used to address a variety of infections brought on by bacteria, which can be multidrug resistant. These infections are common, which raises treatment resistance and impedes the research and development of new drugs . Analysiotential . The Cyd-amylase . The ant0 to 3 h ,43. ThesCydonia oblonga was collected from Allahdand Dheri , Malakand Khyber Pakhtunkhwa (KP), Pakistan. The taxonomic identity of the plant was authenticated by a botanist in the Department of the Botany University of Swabi.The bark of Gallic acid and quercetin were purchased from Sigma-Aldrich France, and nutrient agar was obtained from Oxoid Ltd. England. 2,2-Diphenyle-1 picrylhydrazyl (DPPH) and Folin–Ciocalteu reagent , aluminum chloride, methanol, iron chloride, sodium hydroxide, sodium nitrite, and ascorbic acid were purchased from Sigma-Aldrich, Germany. All the chemicals utilized in assays were of analytical grade, except HPLC-grade solvents, which were purchased from Dae-Jung, Korea. They were used as such without any further purification.Cydonia oblonga was washed and dried under a hood to avoid deterioration of bioactive active compounds due to sunlight. The dried sample was ground into a powder and mixed well with methanol. After three days, the extract was filtered through Whatman filter paper, and using a rotary evaporator  the solvent was evaporated from the filtrate under reduced pressure and at 40 °C. HPLC analysis of the crude was carried out using a reported protocol [The fresh bark of the protocol . Then, 7Cydonia oblonga bark using reported assays [Preliminary phytochemical screening was undertaken to determine the presence of various bioactive constituents such as alkaloids, flavonoids, glycosides, terpenoids, and tannins in Met + Ext of d assays ,46,47.Cydonia oblonga bark Met + Ext and purified fractions from F1 to F7 using Folin–Ciocalteu assay [3 (sodium carbonate) solution was successively added to the mixture. The mixture was made up to 25 mL volume with distilled water and thoroughly mixed. After 90 min, the absorbance was measured at 760 nm using a UV spectrophotometer. Gallic acid solution curve (0 to 100 mg/mL) was used as a standard. Total phenolic content was expressed as gallic acid equivalents (mg GAE/g) per gram of dry sample, which is a common reference compound.The content of phenolics was determined in eu assay . For the2 (5%), 150 µL AlCl3 (10%), and 200 µL NaOH (1 M). A calibration curve was prepared by using the methanolic solution of quercetin in concentrations ranging from 0–100 μg/mL. The resulting mixture was incubated for 5 min; absorbance was measured spectrophotometrically at 510 nm. The total flavonoid content was expressed in terms of Quercetin equivalent to mg of QE/g of dry samples.The total flavonoid content was determined using the aluminum chloride calorimetry method  with litHPLC analysis was performed according to a previously reported protocol  with somv/v) and solvent B . The system was conditioned with the mobile phase at least for 30–40 min. The gradient profile started with 100% solvent A at 0 min, 85% A at 5 min, 50% solvent A at 20 min, at 25 min solvent A decreased to 30%, and finally, 100% solvent B at 30–40 min. The solvents were delivered at 1 mL/min flow rate, and the absorbance of phytochemicals was measured at 280 nm wavelength, by using retention times (chromatographic comparisons), and UV spectra were recorded with the UV detector identification of different compounds. Seven active fractions were obtained (F1 to F7), which were further analyzed by GC-MS.The sample was analyzed in an Agilent 1260 infinity High-performance Liquid Chromatography (HPLC) instrument equipped with an autosampler, quaternary pump, degasser, and ultraviolet (UV) detector. An Agilent Zorbax Eclipse C18 column was used for separation. The mobile phase was solvent A  using a previously reported protocol . The sysCydonia oblonga against Gram-positive  and Gram-negative bacteria  using the disc diffusion method. Standard bacterial cultures were used in which the viable count in each culture was determined using a surface viable technique. The bacterial cultures (from 108–109 colony forming units per mL) were swabbed uniformly on the surface of the prepared media using a separate sterile cotton swab each time. Sterilized filter paper discs  impregnated with 20 µL of sample solution were placed individually at equal distances on the surface of seeded agar plates, followed by incubation of bacteria at 37 °C for 24 h in an incubator. Respective solvents without plant extracts were taken as a negative control. After incubation, the results were observed, and we measured the zone diameter around each disc. The experiment was performed in triplicate and mean zone of inhibition values were calculated.In vitro antibacterial activity was examined for Met + Ext and purified fractions of A○ is the absorbance of pure DPPH and B○ is the absorbance of samples. IC50 values were determined using ascorbic acid taken as a standard, prepared with a concentration of 5 mg/5 mL. Serial dilutions were performed with different concentrations of ascorbic acid in the samples at 1000, 500, 250, 125, and 62.5 µg/mL. All the experiments were performed in triplicate.The stock solution was prepared by dissolving 20 mg DPPH in 100 mL of methanol; 3 mL of this solution was taken and its absorbance was measured spectrophotometrically at 517 nm and was considered as a negative control solution. For free radical formation, a stock solution was covered with aluminum foil and incubated at room temperature in dark conditions for 24 h. About 5 mg of extract and each purified fraction were dissolved in 5 mL methanol to obtain the stock solution. Then, the above solution was serially diluted in methanol at the following concentrations: 1000, 500, 250, 125, and 62.5 (µg/mL). Then, 2 mL of each sample solution was mixed with 2 mL solution of DPPH and after reaction for 15 min in dark, the absorbance values of the samples were measured. DPPH radical scavenging activity of plant extract and fractions were calculated by using the following equation:The α-amylase solution was prepared by dissolving 10 mg enzyme in 100 mL distilled water. Then, 10 µL α-amylase enzyme solution was mixed with each test sample in the concentration range of 30 µL. As a negative control, 30 mL of sodium phosphate buffer was added to 10 µL enzyme solution and incubated at 37 °C for 10 min. Then, 40 mL of starch solution was added to each test tube. Incubation was carried out again at 37 °C for 30 min, then 20 µL of 1 M HCL was added and absorbance was measured at 540 nm using a spectrophotometer. The control samples were prepared without any plant samples. Equation (2) was used to estimate the percent inhibition of the enzyme.p-nitro-phenyl-α-D-glucopyranoside (5 Mm) solution in 0.1 M phosphate buffer at pH 6.9 followed by 15 min incubation at 37 °C. The absorbance of the final reaction mixture was recorded at 405 nm. The reaction mixture with no plant extract was used as positive control while the blank solution was prepared without the enzyme α-glucosidase. The α-glucosidase % inhibition was calculated using the formula:The reaction mixture was formulated by adding 100 µL of α-glucosidase enzyme (0.5 units/mL), 0.1 M phosphate buffer (600 µL) at pH 6.9 and 50 µL of each sample dilution . The mixture was incubated for 15 min at 37 °C. The enzymatic reaction began by adding 100 µL 50 for % DPPH, α-amylase, and α-glucosidase inhibition, Excel 2007 was used. The standard deviation and means were estimated using Excel.To calculate ICCydonia oblonga has numerous biologically active compounds such as polyphenols and flavonoids. The crude extract and fractions isolated through HPLC were subjected to GC-MS analysis. A total of twelve compounds were identified through HPLC and GC-MS analysis. The extract and various fractions exhibited potential biological activity such as antioxidant, antibacterial, and anti-diabetic activities. This bark can constitute an alternative therapeutic agent for bacterial infections, oxidative stress, and diabetes, which are major subjects for future research. Pharmacological and toxicological studies are also needed to confirm the hypothesis tested here, which will also further elaborate on the safety profile of the extract.HPLC analysis revealed that the bark of