Here we present anewly synthesized bifunctional organic chromophorewith appealing spectroscopic and nonlinear optical features. The positionsof absorption and emission maxima of the dye vary with increasingsolvent polarity and exhibit positive solvatochromism. The determinedchange in the dipole moment upon excitation based on the Bilot andKawski theory is 5.94 D, which corresponds to the intermolecular displacementof a charge equal to 1.24 Å. An investigated organic-based systemrepresents a significant, repeatable, and stable over time opticalsignal modulation in the manner of the refractive index value. Itsmagnitude is varied both by optical pumping intensity as well as byexternal frequency modulation, which indicates that such system isan alluring and alternative core unit for optoelectronic devices andcomplex networks. Then, the same active system, due to the nonresonantmechanism of higher harmonics of light inducement, can provide secondand third harmonic signals. According to the introduced laserline spatial modifications , it is resulted in output SHG signal with magnitude variedabout 100%. Its magnitude is noticeably small; however, to constructsensitive optical sensors or infrared indicators, such feature mayguarantee satisfying circumstances. On the other hand, multifunctionalityand versatility are the most desired molecular properties. Indeed,there are research fields, like photonics,6 optoelectronics,7 spectroscopy,9 medicine,10 and industrial areas, such as complex networks,11 optical databases,13 and logic and/or optical gates,14 whereremote-controlled materials, especially those driven by light, arethe most appealing ones. According to the reasons listed above, thereare already few different approaches for designing such working organicsystems, starting from organic low-molecular units embedded into branchedand complex matrices15 and passing by nano-object utilizationin both organic/inorganic systems16 upto the macromolecular, self-organizing, and functional systems.17 Understandably, each of them has advantagesbut also has drawbacks.In the last few decades,there has been a particular interest inorganic, multifunctional materials.20 An active part is typically composedof a small chromophore, which is responsible for matter vs light interaction,provides luminescence and light amplification phenomena,18 or modulates the output signal according tothe particular mechanisms, i.e., by multiple photoinduced isomerizations.21 Sometimes, it is even possible to observe nonresonantinteractions with strong coherent light generating in this way higherharmonics of light, such as second or third harmonic .23 When focusing on the macromoleculararchitecture, usually it plays the role of stabilizing the whole system.21 In particular, the polymeric matrix is opticallypassive and transparent, which enables photons to penetrate its volumeeasily and reach active molecules. Furthermore, frequently, thanksto the particular branched structure, the polymer provides molecularhooking or hindrance for low-molecular active compounds (which dependson the available amount of free volume).21 Semi-intercalated and photoresponsive materials can follow variousprocesses responsible for the aforementioned physical phenomena .When considering the first mentionedlow-molecular organic materialsembedded in more sophisticated matrices, we need to discuss typicalguest–host systems.Here,we present an oxazolone derivative, abbreviated as Ox-π,π-Ph(OMe),which represents a delightful example of a multifunctional photoresponsivematerial. We have experimentally proved and theoretically supportedthe ability to generate two stable and photoinduced conformers responsiblefor effective refractive index changes. Such modification resultsin two optical features: its isotropic  and anisotropic(light controlled) states. Such signal’s optical modulationrealized in a range of seconds (static) and microseconds (dynamicmode) allows for easy and effective optical data manipulation. Moreover,by implementing a high-power picosecond laser system, it was ableto generate significant SHG and THG signals, whose magnitude was alsotunable according to the implemented various rotation angles. Thus,we believe that such material’s development can pave the wayto construct optoelectronic or spectroscopic highly responsive andeffective organic-based devices.H)-one reveals the desired nonlinear optical propertiesto serve as a productive light amplifier, generator of higher harmonicsof light, and optical modulator in the sub-microsecond time scale.24 Therefore, we decided to modify its structure to obtain an oxazolonedye with desired properties for various applications in spectroscopy,photonics, and optoelectronics. Since one of the most widely appliedmethods for adjusting the photophysical properties of organic compoundsis to play with the nature of the substituents,27 we have synthesizedan oxazolone dye containing a methoxy group at the ortho position.We hope that the modification influences the electronic distributionwithin the molecule since the compound reveals the D-π-A withthe 5-(4H)-oxazolone ring constituting an electron-withdrawingmoiety and the phenyl ring and methoxy substituent being an electron-donatingpart. Additionally, such configuration of the molecule offers thepossibility of isomerization or molecular rotation.During ourongoing work toward the compounds for optoelectronics, we have demonstratedthat the 4-(3′-phenyl-2′-propenylidene)-phenylooxazol-5(4H)-one ) was synthesized in amoderate yield of 41% from hippuric acid and 2-methoxycinnamaldehydeas shown in Supporting Information).The 4-(3′-phenyl-2′-propenylidene)-phenylooxazol-5 and water, respectively. The estimated E → Z isomerization barrier in GP is slightlymore than 10 kcal/mol with a simultaneous increase to 11.43 kcal/molin the water environment  geometryof the dye in SCT). The most important selected geometry parameters forthese conformers  are listedin Tables S2 and S3 in the Supporting Information. E and Z conformers in GP, TMP, and water as indicators of the geometricalperturbations. In the other solvents tested, the same bonds were shorteror longer regardless of E-Z isomerization.The analysis of structural parameters revealed a highsensitivityof bond lengths and dihedral angles of Ox-π,π-Ph(OMe)to the environmental changes, as well as to those occurring duringphotoexcitation to the first singlet excited state .Changes in their spectral position may indicate the conversion ofthe E isomer to the Z one or thenonspecific interaction with the solvent, i.e., the formation of hydrogenbonds between the solute and solvent molecules.28In line with the theoretical data, the experimentalresults showedthat the dye is not stable in a dilute solution. After 1 week of storingthe dye in ethanol at room temperature, the absorption and fluorescencespectra are blue shifted by approximately 77 and 83 nm, respectively dye, whereasthe basic photophysical data obtained for the tested dye in solventsof different polarity are collected in 24 The mainabsorption band is located at ∼393–412 nm and, in nonpolaraprotic solvents, exhibits a distinct fine vibration structure, whichbecomes almost structureless in a polar environment. The molar extinctioncoefficient has a large value (ε ∼ 3.82 × 104–6.25 × 104 M–1 cm–1) and is lower in polar solvents, although there isno clear correlation between its values and the solvent polarity.The excitation wavelength has no effect on the dye fluorescence spectra,which retain the vibronic structure in a nonpolar environment. However,as the solvent polarity increases, they become structureless and arered-shifted .The bathochromic shift of the fluorescence band is ca. 68 nm in aproticsolvents and only reaches 24 nm in alcohols, possibly due to the hydrogenbonding ability of the solvents, which stabilizes the electronic statesof the dye in a different way. The introduction of an electron-donatingsubstituent to the phenyl ring causes a bathochromic effect comparedto the analogous unsubstituted compound, Ox-π,π-Ph. TheOMe group induces shifts of the absorption and fluorescence maximaof +12–+26 and +24–+54 nm, respectively, depending onthe type of solvent that has a significant influence on their position.24The spectral behavior of the testeddye is similar to the compounddescribed in our previous work.Tables S4–S6). They are discussed in the Supporting Information.To assign the bands observed in the experimentalabsorption spectraand characterize the nature of the electronic transitions, theoreticalspectral properties were determined  state with opposingcharge and bond localization relative to the ground state (GS). Inaddition, the decrease in FQY is related to vibrations and rotationsaround the single and double bonds separating the oxazolone scaffoldfrom the side phenyl rings, responsible for the effective deactivationof the excited state by nonradiative processes. Moreover, in the proticsolvents according to the work of Yang et al.,30 the reduction in FQY is associated with a hydrogen-bonding-inducedquenching of fluorescence.The testeddye has a slightly lower value of the fluorescence quantumyield, ϕxazolone 1. These Table S7 in the Supporting Information). The main lifetime values are slightlyreduced in a more polar environment with a concomitant increase ofthe longer component so that the average lifetimes rise with increasingsolvent polarity from 0.16 ns in n-hexane to 2.81ns in DMSO (Table S7 in the SupportingInformation). Hence, the rate constant of radiative kr deactivation decreases from 12.69 × 106 to 0.16 × 106 s–1, respectively,which is caused by the more polar nature of the solvent (Table S8 in the Supporting Information). Thereis no clear correlation between the fluorescence lifetimes and thedielectric properties of the solvents. Nevertheless, there is a downwardand upward trend when considering the picosecond and nanosecond components,respectively.Fluorescence decays of Ox-π,π-Ph(OMe)in all solventsare at least biexponential. The determined fluorescence lifetimesconsist of a shorter major component of ca. 100–200 ps anda longer one on the nanosecond scale  state. The observed increase of the averagefluorescence lifetimes in more polar environment may result from themore forbidden feature of the CT emission. In addition, as shown in 34 i.e., polarizability(SP), dipolarity (SdP), acidity (SA), and basicity (SB).36 The best correlation results obtained by The effect of the solventproperties on the absorption and fluorescencemaximum shift, as well as the Stokes shift, was estimated based onthe multiple regression analysis using Catalán’s solventpolarity parameters,24 The solvent polarizability plays the most important rolein the solvation of the solute with respect to absorption as indicatedby the percentage of this polarity parameter. Since the absorptionband position is mainly controlled by nonspecific interactions, theabsorption arises from the polarized π–π* transition.21 At the same time, the negative sign indicatesthat the absorption band shifts toward longer wavelengths with increasingpolarizability (SP), dipolarity (SdP), acidity (SA), and basicity(SB) of the environment. On the other hand, fluorescence is the mostsensitive to changes in the polarizability (SP) and dipolarity (SdP)of the solvents. Its maximum is red-shifted in more polar solventsdue to the high ratio between SP and SdP, although a hypsochromicshift is observed with respect to SA and SB, accordingly. However,the latter terms are negligible in fluorescence analysis since theyare characterized by the lowest values and high standard errors. Inaddition, an excitation led to changes in SA and SB terms, which indicatethe lower acidity and basicity of the excited state37 of the Ox-π,π-Ph(OMe) dye.To compare the data obtained basedon multiparameter linear regression,the percentages of individual solvent polarity parameters were determined.According to the results, the Ox-π,π-Ph(OMe) dye behavesin terms of absorption, fluorescence, and Stokes shift in the solventsused similarly to the compound described previously.–1 with the solvent type, i.e.,from 4431 cm–1 in MCH to 6441 cm–1 in MeCN and from 4548 cm–1 in 1-BuOH to 5707 cm–1 in MeOH, respectively. The increase in the Stokesshift in different environments is due to the increase in solventdipolarity. The effect may relate to the separation of charges inthe D-π-A dye molecule and creation of a dipolar structure differentlyoriented in space as observed in other compounds with a donor–acceptorsubstituent displaying a low or moderate effect.38Followingthe experimental data, the Stokes shift changes by approximately2010 and 1149 cmE and Z) corresponds basically to the HOMO → LUMO transition excitation for both conformers (ansition5. HOMO eEGAP). The ΔEGAPE – Z difference is 0.44 eV in the gas phase, and it isincreased to 0.49 eV in the aqueous phase (Table S9 in the Supporting Information).There is also no significant change in the value of the energyseparation between HOMO → LUMO orbitals (r)) calculatedfor the first electronic transition is graphicallydepicted in Figure S5. The Δρ(r) plots show that the density depletion zones (blue) aremostly delocalized on the benzene rings. The regions of density increment(purple) are mostly localized on the carbon bridge and oxazolone ring.At the same time, the solvent polarity does not significantly changein the location of these regions. This is reflected in the amountof transferred charge (qCT) and charge-transferdistance (DCT). Both qCT and DCT values are largefor the Z isomer. The ΔρCTZ – E is 0.1 a.u. (Table S10 in the Supporting Information), while the ΔDCTZ – E is 0.3 Å in lesspolar solvents and 0.05 Å in more polar solvents. Regardlessof the isomer type, the DCT indicatesthe CT character and confirms the contributions from HOMO →LUMO transition, although minor contributions from other orbitalsshould be expected .The density variationupon photoexcitation (Δρ(ab) andfluorescence (νfl) band shift of a spherical solutein solvents with different permittivity (ε) and refractive index(n), based on eqs S1 and S2 (see the Supporting Information), which result from thetheory of Bilot and Kawski43 by employing the quantum–mechanical perturbation theory,give straight lines with the correlation coefficients being largerthan 0.98, which indicate a good linearity for both m1 and m2  are ratherhigh, indicating a significant variation of the dipole moment uponexcitation. This suggests that the emission of the Ox-π,π-Ph(OMe)dye originates from a state that is more polar than the ground state.Correlations between the absorption  using eqs S5, S6, and S7, we got μTable S11. First, the dipole moment value in the ground (μGS) and excited (μCT) state increases as a functionof solvent polarity for both E and Z isomers. The E isomer is characterized by a highervalue of the CT excited-state dipole moment relative to the Z isomer (ΔμGSE – Z: 0.97 → 3.69 D from vacuum to water). However, forthe Z isomer, a higher μGS is observedrelative to E (ΔμGSZ – E: 0.88 → 1.02 D). At the same time, both isomersare characterized by a high polarity of the excited state (ΔμCT – GS), and the ΔμCT – GS exceeds 6 D. Moreover, although the vector orientation is similarin both conformers  behavior of organiccompounds. Such molecules typically have a π-conjugated framework,end-capped by electron-accepting and electron-donating groups to enhancethe polarizability and hyperpolarizability.49According to the literature,E/Z conformers are collected in Supporting Information. For both E and Z conformers, the ⟨α⟩,Δα, and βvec values increase monotonouslywith the solvent polarity. The E isomer is characterizedby higher linear polarizability and first-order hyperpolarizabilityvalues. These observations suggest and at the same time confirm thatthe presence of the methoxy group is a factor maximizing the nonlinearresponse of the compound and the E conformation canbe more efficient in the second harmonic generation (SHG) phenomenon.The polarizability and hyperpolarizability of the moleculeirradiated with intense laser light giving the electric field arethe subject of many studies in terms of understanding various linearand NLO properties. In particular, these studies include the interrelationshipof the nonlinear optical properties with the electronic structureto design new multifunctional molecules. The calculated values for In 51 From the same relation, a nonlinear refractive index (n2) value can be extracted. In the case of the Ox-π,π-Ph(OMe)NLO chromophore, it is equal to (3.92 ± 0.01) × 10–10 cm2/mW  × 10–8 m2/W). The experimental points taken into account for the correlationshown in n kinetics measurementpresented in n was shown as a signal increase , and after the moment, when the darkness, thermodynamicconditions were present (UV light in the ″OFF″ state)and the signal started to decrease. Two approaches were utilized toapproximate experimental curves (Δn inducementand vanishing) as mono- and biexponential by graphical and numericalmethodologies, respectively. The first mentioned time constant valuesof increase and decrease fragments were defined to be 70.0 and 41.3s, respectively. It means that thermodynamic relaxation is an almosttwo times faster process than that artificially caused by UV lightphotoinduced birefringence. The other tendency is shown by time constantvalues estimated by the numerical method. Namely, the very first momentof both UV light irradiation as well as its deactivation is fast (τinc1 = 11.3 s and τdec1 = 12.5 s), whereasthe second part of the same processes is much slower by about 2 ordersof magnitude, giving the following numbers: τinc2 = 178.9 s and τdec2 = 166.8 s, respectively. Typically,in the observed optical Kerr effect, the monoexponential approximationfunction is used:51 however, in the case of the Ox-π,π-Ph(OMe)NLO chromophore, the rule may be different for a structural reason,namely, an existence of two π-bonds localized in the centralpart of the molecule. One of them is partially blocked by the closeheteroatomic ring neighborhood, while the second one is surroundedjust by hydrogen atoms, providing a much higher flexibility in thisregion , which demonstratesa highly stable and responsive organic system controlled fully bylight .In sregion 1.Thus, 51 the macroscopic opticalanisotropy generation originates from multiple E-Z-E (trans–cis–trans) isomerization cycles, which werealready confirmed by quantum chemical calculations results (P = cos2(α), where P meansthe probability of photon absorption and α denotes the anglebetween the polarization direction of the provided laser light andlong axis of the molecule . Therefore, the absorption as well asphotoisomerization cycles will take place up to the moment when opticalanisotropy achieves a photostationary state. Namely, the maximum populationof molecules will be oriented perpendicularly toward applied linearlypolarized UV light. In Ipump = 17.1 mW/cm2) and externalsignal modulation (f = 10 Hz) at the same time. Inthat way, it was possible to show what is the dynamic component’s(multiple E-Z-E isomerization) participation to thetotal Δn value. The total (static) photoinducedbirefringence is marked in yellow, whereas its dynamic component isshown in violet color in Based on the theory ofthe all-optical switching phenomenon observedin the optical Kerr effect, results 5. All thn signal amplitude increases anddecreases, respectively, for selected frequency modulations in therange of 10–175 Hz. It is clearly shown that the needed time(∼110 ms) and amplitude magnitude (2.2 × 10–6) are the highest for the lowest value of external signal modulation(10 Hz). Such tendency is exponentially decreasing along the higher-frequencymodulations up to almost 200 Hz, which is presented in –6 m/V2. Finally, the third-order nonlinear optical susceptibilityvalue was determined using all experimental achievements and led usto define χ(3) as 2.18 × 10–10 in the SI unit system (2.55 × 10–8 cm2/W in cgs).Afterward, the dynamic part of estimated photoinduced birefringencecharacteristics is presented in 54 Going into details, the obtained photoinduced birefringencefor the Ox-π,π-Ph(OMe) NLO active system is in the sameorder of magnitude as its previously investigated derivative . The cited organic system characterizesa Δn value equal to 5.3 × 10–4 as the reached maximum for Ipump ∼50 mW/cm2 and about 3.5 × 10–4 when Ipump ∼ 10 mW/cm224 . Subsequently, other organic NLO dyes, likepyrazolone,21 pyrazoline,52 or thiophene53 derivatives,represent a Δn parameter on the similar levelof photoinduced birefringence, i.e., equal to 5.5 × 10–4, 1.0 × 10–4, and 1.5 × 10–4, respectively. However, in all these cases, the pump intensity usedto achieve such magnitude of photoinduced values was much higher thanin the current situation (or in pyrazolone or oxazolone derivatives).Meanwhile, if comparing azobenzene derivatives like DR1, CPND-5, SR7B,or S3 NLO chromophores, Δn values are rathervaried (from 1.4 × 10–3 up to the 4.0 ×10–3).54 All estimatedNLO parameters compared with available literature data are presentedin By making a comparison with other, similarlow-molecular NLO chromophoresexisting in polymeric systems, it can be stated that the presentedresults are in accordance with already achieved developments.56 (marked as the red curve in elec.(3) componentvalue defined for the Ox-π,π-Ph(OMe) NLO chromophore was(3.38 ± 0.02) × 10–22 m2/V2. The same parameter was already estimated for the referencematerial and is equal to 2.0 × 10–22 m2/V2.58 If comparing similarchemical structures for which the third-order NLO susceptibility parameterwas defined, the current results place the Ox-π,π-Ph(OMe)system among the active and significant higher harmonic of light (THG)generators. For instance, the oxazolone derivative cited before ischaracterized by χelec.(3) equal to 6.0 × 10–22 m2/V2,24 whichrepresents the same order of magnitude of the experimental achievement.However, there are known systems where the χelec.(3) parameter is even higher59 or lower  parameter less than 0.5 × 10–22 m2/V2).60 Such observation makes materials engineering even moreattractive and useful because based on the chemical structure pattern,it is possible to influence straightforwardly of the output physical,in particular, spectroscopic properties of the compounds., the SHG magnitude was higher than that in the P one  byabout 1 order of magnitude ((2) value is almostnegligible. The second-order NLO susceptibility value was definedas 0.025 ± 0.001 and 0.056 ± 0.001 pm/V for S and P experimental setup configurations, respectively.Together with the already defined χ(2) parameterfor the quartz slab used as the reference material, which is 1.00pm/V,61 it seems that the Ox-π,π-Ph(OMe)NLO active medium can serve as a very precise SHG sensor that is alsosensitive in a spatial manner.63 When discussing the obtainedχ(2) parameter for the investigated system togetherwith the already known pyrazolone-based NLO system, it is quite similarin generating a low-value SHG signal; however, its magnitude is significantlydifferent and higher (doubled) for the P polarizationconfiguration than the S one ((2) in the P configuration is even 1 order of magnitude higher thanthat in the S one , namely, DCNP or PY-pNO2 molecules64 or disperse red 1.65In agnitude 10c,d. Thhe S one 5. Anyhowelec.(3) value far from 0). Moreover, by applying the appropriatethermoelectrical prefabrication process, it was feasible to breakthe internal symmetry and finally induce the SHG signal, and it wasalso sensitive to spatial changes applied in the experimental setupenvironment. Then, by means of implementing low-energy CW laser emission,it was possible to modulate the refractive index value effortlessly.Using a typical pump-probe setup, we have proven that the Ox-π,π-Ph(OMe)NLO chromophore can characterize both the optical isotropy and anisotropyof the n coefficient, which paved the way to constructlogical gates working on 0–1 modules. Especially, the remote-controlledtwo-distinguishable-states switching process is stable over time withthe same signal amplitude. Such revelations show undoubtedly thatoxazolone derivatives can play a pivotal role as useful and efficientspectroscopy tools for various applications.Inthis contribution, we have shown a chemically modified oxazolonedye with appealing optical and nonlinear optical properties, whichcan be easily implemented into spectroscopy and electro-optical devices.Starting from synthesis, we have presented the ease of fabricationand further manipulation, which are desirable from the application’spoint of view. Quantum chemical calculations confirmed the existenceof two stable, spatially different forms, which are responsible forsignificantly different spectroscopic responses. The internal electroncharge transfer proven by a theoretical approach confirms the efficientmovement of the negative carriers, which causes a significant THGsignal (χTable S1 in the Supplementary Information), werepurchased from either Aldrich Chemical Co. or Chemat Co. Poland, andthey were used as obtained.The reagents for the synthesis,i.e., hippuric acid, 2-methoxycinnamaldehyde, sodium acetate (anhyd.),and acetic anhydride, as well as all solvents ,2-methoxycinnamaldehyde ,sodium acetate , and acetic anhydride .The oxazolone dye, 4-[3-(2-methoxyphenyl)prop-2-enylidene]-2-phenyl-1,3-oxazol-5-one,was synthesized by the method described in the literature1) in acetic anhydride was heated at50–60 °C until a clear yellow solution of compound 1a was obtained (30 min). Then, 2-methoxycinnamaldehyde (2) and anhydrous sodium acetate were added to the solution.The reaction mixture was refluxed with stirring for 4 h. After coolingto room temperature, the mixture was diluted with anhydrous ethanol and kept below 0 °C for 20 min.The precipitate was filtered off under suction, washed with cooledethanol and then with hot distilled water, and dried. The crude productwas purified by flash chromatography using chloroform as the eluentto give an orange crystalline solid. Yield 41% (0.35 g), mp: 151–152°C, TOF MS (ESI) m/z: [M +H]+ calcd for C19H16NO3 m/e 306.1130 (100%), 307.1163 (20.5%), 308.1197 (2.0%); found 306.1129,307.1155, 308.1197.Hippuric acid  δ 8.09–8.07 ,7.72 , 7.68–7.65 , 7.64–7.62 , 7.61 , 7.42 ,7.33–7.30 ,7.13–7.11 , 7.04 , 3.91 ;13C{1H} NMR  δ166.6, 161.5, 158.2, 139.9, 133.5, 134.0, 133.8,132.2, 129.8, 128.7, 128.2, 125.8, 124.7, 124.2, 121.4, 112.3, 56.2;15N NMR  δ 238.07.–1) 3019, 2985,2837, 1781, 1645,1592, 1555, 1489, 1468, 1451, 1358, 1327, 1297, 1254, 1162, 1027,976, 876, 777, 762, 745, 696.IR . Tetramethylsilane (TMS) was used asan internal standard for calibrating the chemical shift. The measurementswere performed in dimethylsulfoxide (DMSO-d6). The IR spectra were recorded on a Bruker Vector 22 FT-IR spectrophotometer(Germany) in the range of 400–4500 cm–1 bythe KBr pellet technique. Melting point (m.p.) values were determinedon a Boëthius apparatus  and werenot calibrated. The TOF MS spectrum was recorded on a Waters LCT PremierXE mass spectrometer with ESI ionization. The HPLC analysis was doneby a Waters Breeze 2 HPLC system equipped with a UV–vis detector(detection wavelength was 420 nm), binary HPLC pump, and a SymmetryC18 column . Separation was conductedunder isocratic conditions with 1.0 mL/min flow rate at r.t., 10 μLinjection volume, and HPLC-grade acetonitrile as a mobile phase.The 1D and 2D NMR spectra wererecorded on a Bruker Ascend 400 spectrometer with a resolution of400 and 100 MHz for –5 and 10–6 M for absorptionand fluorescence measurements, respectively.The UV–vis absorption spectra from solutions were recordedat room temperature on a UV–vis Multispec-1501 spectrophotometerfrom Shimadzu, whereas the fluorescence spectra were determined usingan F-7100 spectrometer from Hitachi. The solution concentration wasca. 10av, was calculated as τav = (Σiαiτi)/(Σiαi), where αi and τi are the amplitudes andlifetimes, accordingly. The compounds were studied at the concentrationneeded to provide an absorbance of 0.2–0.3 in a 10 mm cellat 375 nm.The fluorescencedecay curves were recorded on a single-photoncounting system (FLS920P Spectrometer) from Edinburgh Instruments.The sample was excited by a picosecond diode pulsed laser source witha high repetition rate (pulse ca. 55 ps at 375 nm). Photons emittedby the sample were detected with a fast microchannel plate photodetector,and the time with respect to the excitation pulse was measured. Thefluorescence decay curves were analyzed using the Fast program, andthey were fitted as sums of two exponentials. The average lifetime,τref = 0.3868) as reference. The absorbance(A) of both the dye and reference solution at anexcitation wavelength (380–404 nm) was ca. 0.1.The fluorescence quantum yield (FQY) was calculatedby the comparativemethod using Coumarin 153 in an ethanol solvent  and theBakhshiev69 as well as Bilot and Kawski42 theories including the Onsager description of nonspecific electrostaticsolute–solvent interactions.The solventeffect on the spectral properties of the tested dyewas analyzed by applying multilinear correlation based on the four-parameterCatalánSGS) and excited states (SCT) werecalculated using the density functional theory (DFT) approach implementedin the Gaussian 09 program package70 withthe TIGHT threshold option and PBE0/6-311++G basis set. To verifythat all the structures correspond to the minima on the potentialenergy surface, an analysis of Hessians was performed. The electronicproperties were characterized by computations of the vertical absorptionand emission spectra, which were obtained using the time-dependentdensity functional theory (TDDFT/PBE0)71 and by including the state-specific (SS) corrected linear response(cLR) approach.72 All spectroscopic calculationswere performed using several different functionals, namely, the standard-hybridPBE074 functionals. The dipole moments and polaritiesof the charge-transfer state (CT) were evaluated by numerical differentiationof the excitation energies (E) in the presence ofan electric field F of 0.001 a.u. strength:75iF corresponds tothe electric field applied along the Cartesiandirection and i and μi are the ith Cartesian component of the electricdipole moment, respectively. The isotropic average polarizability(⟨α⟩), polarizability anisotropy (Δα),and first-order hyperpolarizability (βvec) were determinedbased on the Gaussian 09 program and were defined as follows:i is given by All geometrical parameters ofthe investigated molecules in their ground (DCT) and the amount of transferred charge (qCT) have been determined following Le Bahers’sprocedure.76 The solvent effect on thelinear and nonlinear optical properties has been taken into accountusing the Integral Equation Formalism for the Polarizable ContinuumModel (IEF-PCM).78The density differences were obtainedat the PBE0/6-311++G level represented with a contour thresholdof 0.02 a.u. The charge-transfer distance dye) and host (poly(methyl methacrylate), PMMA) were dissolved indichloromethane (DCM). When the solution was homogeneous, it was depositedon the pure glass substrate using the spin-coating  or drop-casting technique  depending on the dedicated experimentalneeds. After complete solvent evaporation, such prepared thin filmswere ready for further studies.Absorption spectra of the thinfilms were collected using a JASCO V-670 spectrometer, whereas theirfluorescence spectra were recorded with the help of Fluoromax-4 HORIBA.Thickness values of each thin film were measured in several randomlyselected points on the sample surface but localized mainly in thecentral part, where all kinds of laser spectroscopy measurements wereperformed. In here, a DEKTAK 3 profilometer was used.51 Here are just some important details. Briefly, the utilized laserlines (pump and reference ones) intercrossed on the sample surface,creating a light spot with a 3 mm diameter. The thin film was localizedbetween two polarizers, turned to each other by 90°. The NLOresponse was read out using a silicon photodiode mounted behind across-polarizer system and prevented by a red filter, which cut offthe pumping laser line. Additionally, to maximize the observed effect,the linearly polarized UV light was oriented to be turned by 45°with respect to the polarization direction of the reference beam.51 For the optical Kerr effect investigation, the measured and averagedsample thickness was 24.5 μm.The opticalKerr effect (OKE) phenomenon was investigated in atypical pump-probe laser setup. The diode pumped solid-state (DPSS)lasers with emission at 405 and 638 nm were introduced as pump andprobe beams, respectively. All principles defining the OKE experimentand comprehensive characterization of the third-order NLO effects are described in the literature.79 the second-order NLO susceptibility value (χ(2)) and electronic component of χelec.(3) were estimated. The experimentalsetup describing introduced electro-optical elements is describedin the literature;80 however, several importantdetails will be provided also in this contribution. The picosecondpulsed laser source was utilized to provide the fundamental beam witha repetition rate equal to 10 Hz (t = 30 ps) andgenerated wavelength at 1064 nm. Input and output signals were monitoredby electro-optical controllers. The initial beam was illuminatingthe sample surface, which was rotated from −60/–80°up to +60/+80° depending on the observed effect and type of sample. For the THG experiments,a spin-coated sample was used (dTHG =1.72 μm), whereas for the SHG ones, a drop-casted layer wasprepared (dSHG = 13.11 μm). Suchapproach was motivated by a particular nature of the performed experiments.Since the investigated Ox-π,π-Ph(OMe) NLO chromophorecharacterizes centrosymmetry, it was necessary to use an additionalprocedure for breaking the symmetry, namely, the corona poling technique.82 For a more efficient molecular ordering using thermoelectric methods,the used sample thickness was increased upon above of 10 μm.If considering the second harmonic generation investigation, as itwas mentioned before, the drop-casted layer was transformed accordingto the corona poling procedure. The sample was treated by a highertemperature (100 °C) and applied DC voltage (8 kV) for 10 min.After that time, with the still applied external electric field, thethin film decreased its temperature and sustained its broken symmetry,which enabled further SHG studies. To analyze experimental resultson both THG and SHG experiments, the Kubodera and Kobayashi56 as well as Lee83 theoreticalmodels were introduced, respectively.To achieve a complementaryinvestigation of nonlinear optical featuresof the Ox-π,π-Ph(OMe) chromophore, the ability of higherharmonics of light generation was experimentally checked. Using acomparable technique based on the Maker fringe measurements,


Photosystem I (PSI) with its associated light-harvesting system is the most important generator of reducing power in photosynthesis. The PSI core complex is highly conserved, whereas peripheral subunits as well as light-harvesting proteins (LHCI) reveal a dynamic plasticity. Moreover, in green alga, PSI–LHCI complexes are found as monomers, dimers, and state transition complexes, where two LHCII trimers are associated. Herein, we show light-dependent phosphorylation of PSI subunits PsaG and PsaH as well as Lhca6. Potential consequences of the dynamic phosphorylation of PsaG and PsaH are structurally analyzed and discussed in regard to the formation of the monomeric, dimeric, and LHCII-associated PSI–LHCI complexes. Light-induced primary charge separation occurs between the primary electron donor (P700) and acceptor (A0). The reduced A0 donates an electron to iron–sulfur cluster FX via phylloquinone (A1). These redox components as well as most antenna pigments are located on a heterodimer of the two homologous subunits encoded by the chloroplast psaA and psaB genes. Reduced FX forwards an electron to iron–sulfur cluster FA and subsequently to the other iron–sulfur cluster FB that reduces ferredoxin (FDX) as the terminal electron acceptor of PSI. FDX in turn reduces the FDX-NADP-reductase (FNR), leading to the formation of NADPH. The core of PSI further harbors approximately 100 Chls [Arabidopsis thaliana, an additional Lhca1-a4 dimer was found to be bound on the PsaL side [The core of photosystem I (PSI) is highly conserved from cyanobacteria to vascular plants . The lig100 Chls , which s100 Chls . For exa100 Chls . On the 100 Chls , while P100 Chls . NotablysaL side , suggestChlamydomonas reinhardtii [A. thaliana [C. reinhardtii [C. reinhardtii contains two LHCII trimers (termed as LHCII-1 and LHCII-2) [Binding of LHCII to PSI is a common incidence in most organisms of the green lineage and driven by a process called state transitions . State tnhardtii  or STN7 thaliana . High-rethaliana  and C. rnhardtii   . The LHCLHCII-2) . LhcbM5 LHCII-2) . MoreoveLHCII-2) . These tC. reinhardtii where two copies of Lhca9 tethered two monomeric PSI in a head-to-head fashion, forming a large oligomeric protein complex  be envisioned? Possible key players in the remodeling regulation are likely PsaH and Lhca2. PsaH is an 11-kDa transmembrane protein that is imported into chloroplasts and peripherally associates with the PSI reaction center on the opposite side to the LHCI belt . PsaH knphically . Moreovephically . A recendetected . All thereported . The prethaliana  may indithaliana ,36. Thusnhardtii  or acetythaliana . In C. rtor side . It is pe mutant , which porylated . In the nd Lhca9 ,40. Notand Lhca9 . This inIn the current manuscript, we revisited PsaH and PsaG phosphorylation and reveal that in three independent WT strains, phosphorylation is significantly up-regulated for both proteins. The impact of PsaH and PsaG protein phosphorylation on structure and function of PSI is discussed.C. reinhardtii strains CC-124, CC-125, and CC-4375 IFT46::YFP [−2 s−1 under a 16 h light/8 h dark cycle. One day before the experiment, cells were diluted to 3 µg Chl/ml. Normal light (NL) samples were taken 3 h after the onset of light (NL) and centrifuged for 5 min at 2500× g to pellet the cells. The remaining cultures were diluted to 3 µg Chl/ml and transferred to continuous high light . HL samples were taken after 24 h as described above. The experiment included four replicates per strain and light condition.T46::YFP  were groT46::YFP  on a shag for 10 min to pellet cell debris. Protein concentration in the supernatant was determined by BCA assay . A lysate volume corresponding to 100 µg of protein was tryptically digested according to a modified FASP (Filter-aided sample preparation) protocol [g at room temperature. SDS removal and alkylation of cysteines was achieved by addition of 200 µl of 100 mM Tris-HCl/8 M urea/10 mM TCEP/40 mM chloroacetamide, followed by centrifugation as described above. The detergent removal and alkylation step were repeated three times. Subsequently, samples were washed four times with 200 µl of 100 mM ammonium hydrogencarbonate. Then, trypsin  was added to the filters at a protein-to-enzyme ratio of 50:1. Digestion was carried out overnight at 37°C. Peptides were harvested by centrifugation and trifluoroacetic acid (TFA) was added to a final concentration of 0.5%.To disrupt the cells and extract proteins, lysis buffer (100 mM Tris-HCl (pH 8.5)/1 mM benzamidine/1 mM PMSF/10 mM sodium fluoride/1 mM sodium orthovanadate/10 mM sodium pyrophosphate/10 mM β-glycerophosphate/2% (w/v) SDS) was added to the cell pellets and samples were incubated for 15 min at 65°C in a Thermomixer at 1000 rpm, followed by centrifugation at 20000× protocol . Brieflyg. Membranes were washed twice with 200 µl of 0.1% TFA before peptides were eluted in 100 µl of 60% (v/v) acetonitrile/0.1% TFA. Eluates were briefly mixed and each sample was split into two aliquots: a sample volume corresponding to 10 µg of peptides was used directly for characterization of the proteome (‘nonenriched’), whereas the remaining 90 µg was subjected to phosphopeptide enrichment prior to LC-MS/MS analysis (‘enriched’).Peptides were desalted using C18 membranes (7 mm diameter), cut from Empore C18 extraction membranes (Sigma) using a stencil, and inserted into MobiCol spin columns, sandwiched between two frits . The columns were conditioned and equilibrated using 80% (v/v) acetonitrile/0.1% TFA and 0.1% TFA, respectively. Then, the samples were loaded on the columns followed by centrifugation for 2–5 min at 1000× 2 . TiO2 (1 mg per sample) was activated once with acetonitrile and equilibrated three times with loading buffer  acetonitrile/5% (v/v) TFA/1 M glycolic acid). Then, LB was added to obtain 10% (w/v) TiO2 slurry. Peptide samples were resuspended in 50 µl of LB, then added to 10 µl of TiO2 slurry and the mixture was incubated for 1 h at room temperature at 1200 rpm in a Thermomixer (Eppendorf). Afterward, the slurry was transferred to in-house made C18 STAGE tips . Buffer volume was 80 µl throughout. The buffer was removed and the TiO2 beads were washed twice each with LB and 1% (v/v) acetonitrile/0.1% (v/v) TFA (W1). Nonphosphorylated peptides that bound to the C18 plug were removed by two washes with 80% (v/v) acetonitrile/0.1% (v/v) TFA (W2). Weakly and moderately strongly bound phosphopeptides were eluted from the TiO2 and onto the C18 material by six successive washes with 0.4 M NaH2PO4/1% (v/v) acetonitrile/0.1% TFA. Phosphopeptides were washed once with W1 and subsequently eluted with 60% (v/v) acetonitrile/0.1% (v/v) TFA (fraction 1). Strongly bound phosphopeptides were eluted with 5% ammonia (fraction 2). Phosphopeptides that had detached from TiO2 by the ammonia treatment and bound to the C18 plug despite the high pH were washed once with W1, followed by elution with 60% (v/v) acetonitrile/0.1% (v/v) TFA (fraction 3). All fractions were dried by vacuum centrifugation and stored at –80°C until further use.Phosphopeptides were enriched by metal oxide affinity chromatography (MOAC) using TiO8, Sigma ). The foDried peptide samples were reconstituted in 5 µl of 2% (v/v) acetonitrile/0.05% (v/v) TFA in LC/MS grade water. Samples were analyzed on an LC-MS/MS system consisting of an Ultimate 3000 NanoLC (Thermo Fisher Scientific) coupled via a Nanospray Flex ion source (Thermo Fisher Scientific) to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were concentrated on a trap column  for 3 min using 4% (v/v) acetonitrile/0.05% (v/v) in LC/MS grade water at a flow rate of 10 µl/min. The trap column was operated in back-flush mode, allowing the transfer of peptides on a reversed-phase column  for chromatographic separation. The buffers used were 0.1% (v/v) formic acid in ultrapure water (A) and 0.1% (v/v) formic acid/80% (v/v) acetonitrile in ultrapure water (B). The gradient was programmed as follows: 5% B to 36% over 160 min, 36% to 99% B over 10 min, and 99% B for 20 min. Flow rate was 300 nl/min. The mass spectrometer was operated in data-dependent acquisition mode (‘Top12’), alternating between one MS1 full scan and up to 12 MS2 scans. Full scans were acquired with the following settings: AGC target 3e6, MS1 resolution 70000, maximum injection time 50 ms, scan range: 300–1400 m/z. Settings for MS were: AGC target 5e4, resolution 17500, MS2 maximum injection time 80 ms (200 ms for enriched samples), intensity threshold 1e4, normalized collision energy 27 (HCD)).MS raw data obtained from nonenriched and enriched samples were searched separately with MaxQuant  againsthttp://proteomecentral.proteomexchange.org) via the PRIDE partner repository [Protein LFQ intensity and phosphorylation site intensity data were preprocessed in Perseus : Intenspository  with theChlamydomonas PSI dimer (PDB ID: 7ZQD), PSI-10Lhca (PDB IDs: 6JO5 and 6IJO), and PSI–LHCI–LHCII (PDB ID: 7DZ7).Structural models were generated using PyMOL , UCSF Chimera , ChimerC. reinhardtii grown under NL and HL conditions. Here, our main focus was on PSI phosphorylation. To analyze photosynthetic protein phosphorylation, the algae were cultivated photo-autotrophically in TP medium under a diurnal light regime (16 h light/8 h dark) in NL (60 µmol photons m−2 s−1) for 1 week and then shifted to continuous HL (500 µmol photons m−2 s−1). Samples were taken right before and 24 h after the transfer. After tryptic digestion, peptide samples were split: one aliquot was analyzed directly by LC-MS/MS to obtain information about changes in protein abundances, while another aliquot underwent phosphopeptide enrichment for subsequent phosphoproteomic analysis. To identify differences in protein abundances and phosphorylation that were independent of a genetic background, three C. reinhardtii strains were used in the experiment  and quantitative proteomics data of all strains were combined. In total, 12001 phosphorylation sites from 3337 proteins were identified. After filtering, 2403 quantified phosphorylation sites remained. Differential expression analysis using Limma [We were interested in light-dependent regulatory processes that may affect protein–protein interactions within the photosynthetic protein complexes and therefore compared the phosphoproteomes of ng Limma  (implemeng Limma ) revealeng Limma ,57 and a6f subunit PETO changed in abundance similarly to PSI subunits. The amounts of PETO T161 and S187 residues, which also correspond to a STT7-dependent phosphorylation [Phosphorylation of PSI subunits PsaG  and PsaH (S50) increased from 1.7 to 5.7-fold in response to HL , PSI–LHCI–LHCII , and PSI dimer (PDB ID: 7ZQD), respectively. The PsaG and PsaH subunits are highlighted in different representations. It is of note that the PsaH subunit is present in the state transition complex, while the PsaG is present in all of these structures. Contrastingly, the PsaH has also been observed in PSI–LHCI supercomplex (PDB ID: 6IJO) [The potential impact of increased phosphorylation of PsaG  and PsaH (S50) was further analyzed on a structural level. D: 6IJO) ; howeverSuperposition of PsaL subunits from PSI dimer and PSI–LHCI–LHCII shows a clear tilting of R158 in PsaL subunit of PSI–LHCI–LHCII toward the pS50 in PsaH, thereby implying that this phosphorylation probably aids in stabilizing PsaH subunit in PSI A. Compar6JO5); 143–156 in PSI dimer) in PsaL near the pS50 of PsaH and pT27 of LhcbM1 during state transitions (7DZ7 and 7D0J) but found distorted in the monomeric and dimeric PSI structures. Surprisingly, this loop of PsaL contains the key residues involved in stabilizing the PsaH as well as state transition complexes, such as residues K149 and R154. Though unresolved in the monomeric PSI structures, residue R158 has recently been resolved in the PSI dimer structure (PDB ID: 7ZQD), but it rather points away from pS50 of PsaH, as elaborated above. C. reinhardtii indicate that a stable PSI–LHCI–LHCII complex can exist without phosphorylation of S50. Yet again, there is also a possibility that PsaH S50 could get phosphorylated later when a state II complex has already been formed, thereby further contributing toward the stability of PsaH under this transition. Another possible reason for the phosphorylation of PsaH S50 and stabilization of nearby loop in PsaL could be attributed to the prevention of PSI dimerization under HL, as the structural alignment between PSI–LHCI–LHCII and PSI dimer complexes reveal that they are mutually exclusive, as dimer formation would clash with the physical features of the state transition complex [Moreover, structural alignments indicate the stabilization of a loop .Phosphoproteomic analysis identified the phosphorylation of four residues, namely pS65, pT66, pT71, and pT72, in the loop of PsaG located on the stromal side of PSI. Interestingly, this loop (residues 63–85), together with a short helix  residue of PsaG in the state transition complex, which are 6.4 Å apart from each other. However, the shortest distance between these two residues is 10.9 Å in the PSI dimer. Furthermore, superposition of PsaG from PSI dimer and the state transition complex reveals a change in conformation in some residues near the Lhca1 pole. Such a change is dramatic for the PsaG F63 and D64 , subsequently giving rise to additional interactions with the PsaB subunit in state transition complex. Pi–Alkyl interactions, for instance, could be observed for F63 of PsaG with R301 and A304 of PsaB  which, on the other side, are not evident in the PSI dimer. Interestingly, this is the region where the phosphorylated residues (pS65 and pT66) also occur. It is well-known that phosphorylation has the ability to modify the structural conformation of a protein ,63. TherC. reinhardtii is induced by HL [C. reinhardtii [C. reinhardtii, phosphorylation of PsaH is STT7 independent [Arabidopsis, where closely packed PSI dimers were more abundant in the dark state [C. reinhardtii [Previously, it has been shown that state transition in ed by HL . PsaG phnhardtii , thus inependent  but alsoependent . Due to ependent . In thisependent  and/or aependent . Likewisependent  under HLrk state . In thisnhardtii . Thus, dC. reinhardtii reveal dynamic phosphorylation in response to different light conditions. Structural analysis indicates that the phosphorylation of PsaH subunit at position S50 seems to be important for its stabilization to the PSI, ultimately facilitating the state transition complexes. On the other hand, phosphorylation of PsaG subunit appears to be important for the formation of both PSI dimer and state transition complexes due to stabilization of PsaG and Lhca9 to the PSI. Moreover, besides phosphorylation, stabilization of loops in PsaG and PsaL subunits appear to further aid these processes as they harbor several key residues engaged in either direct interaction with the phosphorylated residues or providing additional interacting partners for the stabilization of subunits necessary for the dimerization of PSI as well as formation of the state transition complexes in green algae.Conclusively, the PsaG and PsaH subunits of PSI in Click here for additional data file.Click here for additional data file.


Here, we analyze endogenous expression of the PRG5 protein in different mouse brain areas, as well as on a subcellular level. We found that native PRG5 is expressed dendritically, and in high abundance in areas characterized by their regenerative capacity, such as the hippocampus and the olfactory bulb. During adult neurogenesis, PRG5 is specifically expressed in a late phase after neuronal cell-fate determination associated with dendritic spine formation. On a subcellular level, we found PRG5 not to be localized at the postsynaptic density, but at the base of the synapse. In addition, we showed that PRG5-induced formation of membrane protrusions is independent from neuronal activity, supporting a possible role in the morphology and stabilization of spines.During adult neurogenesis, neuronal stem cells differentiate into mature neurons that are functionally integrated into the existing network. One hallmark during the late phase of this neurodifferentiation process is the formation of dendritic spines. These morphological specialized structures form the basis of most excitatory synapses in the brain, and are essential for neuronal communication. Additionally, dendritic spines are affected in neurological disorders, such as Alzheimer’s disease or schizophrenia. However, the mechanisms underlying spinogenesis, as well as spine pathologies, are poorly understood. Plasticity-related Gene 5 (PRG5), a neuronal transmembrane protein, has previously been linked to spinogenesis  Complex molecular processes during the development of the central nervous system (CNS) lead to an intricate network of highly specialized cells that communicate via synaptic activity. This development is established by a well-organized progression of events that are still poorly understood. Research has often focused on early embryonic stages of CNS development, but important steps of neuro-differentiation also continue after birth. Postnatal development involves axonal pathfinding and dendrite outgrowth, as well as establishing cell-cell contacts during spinogenesis and synaptogenesis . Even inIn both postnatal development and adult neurogenesis, late neurodifferentiation is characterized by the formation of dendritic spines . These mSo far, several molecules involved in spinogenesis have been identified . One of In vitro analysis of developing hippocampal neurons showed a shift in expression, from an equal distribution along all neurites toward increased localization only along dendrites  mouse-brain sagittal and coronal sections. We detected PRG5 immunofluorescence throughout the brain, but enriched in the hippocampal formation (HPF), cerebellum (CB), and the olfactory bulb (OB) . FluoresIn the cerebellum, (CB) we found that Purkinje cells (pu), marked by parvalbumin (PV), strongly expressed PRG5 in their soma and in their intricately branched dendritic arbors in the molecular layer (mo) . HoweverTyrosine hydroxylase (TH) -positive dopaminergic neurons in the MOB expressed PRG5, as shown by an overlay of fluorescent signals .in vitro results, where PRG5 mRNA expression was restricted to primary cultured neurons and was not found in primary cultured astrocytes  sections for PRG5 expression, by immunostaining with the PRG5 antibody. We found a wide distribution of the PRG5 protein throughout all brain areas, with particularly high abundance in the medial pallidum , the developing hippocampus . NeuronaTo gain further insight into the time point of PRG5 expression during neurodifferentiation, we analyzed its expression during adult neurogenesis. In PRG5 protein abundance was not found during RMS migration of neuroblasts, but was first detected when they entered the olfactory bulb; it increased in radial migrating cells in the olfactory bulb . These fAnother dendritically expressed PRG family member, PRG1, has been shown to be located at the postsynaptic density and to be involved in excitatory synaptic transmission . As synaTo further analyze the localization of PRG5 at the synapse, we conducted immunostaining of murine primary hippocampal neurons. We found PRG5 not to be co-localized to marker proteins of the pre- and postsynaptic transmission sites, bassoon and homer1 , top. InIn PRG5 overexpressing neurons, activity inhibition reduced the number of overall protrusions , Line 24In this study, we analyzed for the first time the expression and localization of the endogenous PRG5 protein in mouse brain tissue. We identified a PRG5 antibody that specifically detects PRG5 protein in mouse brain tissue samples, in both western blot and immunofluorescence experiments. We confirmed a membranous localization of the endogenous PRG5 in the brain, as well as its N-glycosylation, most likely at the predicted glycosylation site at asparagine 158 . In westUsing qRT-PCR, PRGs have been shown to be developmentally regulated on the mRNA level . We analin vitro overexpression studies demonstrated a participation of PRG5 in the formation, morphology, and stabilization of dendritic spines . All protocols were in accordance with the German Animal Protection Law and were approved by the local ethics body of Mecklenburg-Western Pomerania (LALLF) and Lower Saxony . For determining embryonic stages, the day of the vaginal plug following mating was assigned as embryonic day 0.5 (E0.5). Mice of both sexes were used for protein lysis and immunostaining.Time-pregnant, postnatal, and adult wild-type C57BL/6J mice were procured from the central animal facility of the University Medical Center Rostock or the central animal facility of the Carl von Ossietzky University of Oldenburg. They were kept under standard laboratory conditions in accordance with German and European guidelines for the use of laboratory animals  and water 2 and 37°C. Cells were passaged every 3–4 days at around 90% confluency. For transfection, cells were seeded in 100 mm petri dishes with 1.5 × 106 cells per dish and transfected after 24 h with calcium-phosphate precipitation. For this purpose, 22 μg DNA in 337.5 μl sterile H2O and 37.5 μl 2.5 M CaCl2, was precipitated by slow drop-by-drop addition of 337 μl HEPES-buffered saline pH 7.05 , then incubated for 30 min at RT and added to the cells. Cells were lysed after 24–48 h. The following expression plasmids were used for transfection: peGFP-N1-mPRG1, peGFP-N1-rPRG2, peGFP-N1-mPRG3, peGFP-C1-mPRG4, peGFP-N1-rPRG5  were routinely maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine , 100 units/ml penicillin, and 100 μg/ml streptomycin (PAN-Biotech) under standard conditions of 5% (v/v) CON1-rPRG5 .For obtaining protein lysates of different organs and brain areas, mice of varied developmental stages were sacrificed by cervical dislocation, individual organs were removed, and specific brain areas dissected on ice. For embryonic stages, tissue samples of all embryos of one litter  were pooled. Postnatal tissue samples were pooled from four to eight mice of both sexes. For cortex samples, three pooled samples from three independent preparations were used. For other brain areas, one pooled sample of one preparation was used . Liver aCortex tissue was then homogenized with a syringe and cannula in lysis buffer containing 20 mM Tris-HCl pH 7.5, 250 mM sucrose, 1 mM EGTA, 5 mM EDTA, 1x cOmplete mini protease inhibitor cocktail  and 1x PhosSTOP phosphatase inhibitor (Sigma-Aldrich). Cultured cells were washed twice with ice-cold 1x PBS, lysis buffer was added, and cells were scraped off the culture plates with a cell scraper. All samples were sonicated for 3 × 3 s and then incubated on ice for 30 min. Cell debris was pelleted by centrifugation at 10,000–12,000 × g for 10–15 min at 4°C and total protein lysates were stored for the short term at –20°C or the long term at –80°C. For separation of cytosolic and membrane protein fractions, total protein lysates were further centrifuged at 200,000 × g for 30 min at 4°C. The supernatant was collected as the cytosolic protein fraction, while the pellet was re-suspended in lysis buffer additionally containing 1% (v/v) Triton X-100, and incubated for 10 min on ice. Samples were again centrifuged for 10 min at 4°C and 100,000 × g; the resulting supernatant contained fractions of membrane proteins. Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific) with BSA standards, following the manufacturer’s protocol.For purification of eGFP fusion proteins, lysates of transfected HEK293H cells were processed with the μMACS GFP Isolation Kit  according to the manufacturer’s instructions.For the deglycosylation assay, P15-cortex protein lysates were treated with Peptide-N-glycosidase F  as recommended by the manufacturer, run over night at 37°C. Negative controls were incubated without PNGase F.Protein lysates were diluted in 5 × Laemmli buffer containing 1% (v/v) 2-mercaptoethanol or, for PRG5 detection, without 2-mercaptoethanol but with the addition of 8 M urea. Proteins were separated by SDS-PAGE and transferred on nitrocellulose (NC) membranes with tank-blotting procedure for 70 min at 100 V and 4°C. To establish the total protein content, blotted membranes were briefly stained with 0.1% (w/v) Ponceau-S in 5% (v/v) acetic acid. Non-specific antibody binding was blocked with 5% (w/v) bovine serum albumin  or 2.5–5% milk (Carl Roth) in PBS or TBST. Primary antibody incubation was carried out in blocking solution overnight at 4°C  1: 10,000 in 2.5% (w/v) BSA in PBST or TBST for 1.5 h at RT. Membranes were again washed 3 times at RT with PBST, TBS or TBST, and any immunoreaction was detected with Clarity or Clarity Max ECL substrate  according to the manufacturer’s protocol.4 in 0.1 M PB pH 7.2 for 15–60 min at RT, with mild agitation. Slices were washed four times for 5 min with PB, followed by blocking of non-specific antibody binding overnight at 4°C with 10% (v/v) FBS or 1% BSA, 1% (v/v) normal goat serum , and 0.2% (w/v) saponin in PB. Primary antibodies were diluted in 5% (v/v) FBS or 0.5% BSA, 1% (v/v) NGS and 0.2% (w/v) saponin, and slices were incubated for 48 h at 4°C, with gentle agitation. Primary antibodies and concentrations are listed in RRID:SCR_002978) and The Allen Developing Mouse Brain Atlas (RRID:SCR_002990). All immunostainings were performed at least three times with sections from at least three different animals with different sexes. Stainings revealed similar results for all animals and sexes and representative images were chosen for this publication.Adult mice were deeply anesthetized with sodium pentobarbital , then transcardially perfused with cold 1x PB, followed by cold 4% (w/v) paraformaldehyde  in 1x PB fixation solution. P0 mice were sacrificed by cervical dislocation. Brains were removed and additionally immersion-fixed in 4% (w/v) PFA in 1x PB for 24–48 h at 4°C. Coronal or sagittal 35–40 μm brain sections were prepared on a Leica VT1000S vibratome . To reduce autofluorescence, slices were treated with 1% (v/v) NaBH2 in Minimal Essential Medium (Thermo Fisher Scientific) supplemented with 0.6% glucose, 10% (v/v) horse serum, and 100 U/ml penicillin/streptomycin (PAN-Biotech). Media were changed after 3–4 h to Neurobasal A media, supplemented with 2% (v/v) B27, 0.5 mM glutamine  and 100 U/ml penicillin/streptomycin.Hippocampi of all E18 (± 0.5 days) mouse embryos from one pregnant mouse were dissected and primary neuron cultures prepared as previously described by Primary cultured neurons were transfected at DIV 14 with Effectene  according to manufacturer’s instructions. Media was changed 3 h after transfection. The following expression plasmids were used for transfection: pEGFP-N1-rPRG5  and pECF2O; Sigma-Aldrich, A8054), 10 μM nifedipine  and 1 μM tetrodotoxin (TTX)  -2-amino-5-phosphonopentanoic acid (D-APV) (in ddH Israel) .For immunocytochemical staining, neurons on coverslips were fixed with 4% (w/v) PFA and 15% (w/v) sucrose in PBS for 15 min at RT. For stainings including the PRG5 antibody, coverslips were washed three times with PBS and cells were blocked with 5% (v/v) NGS and 0.2% (w/v) saponin in PBS for 1 h at RT. Primary antibody incubation was carried out overnight at 4°C in 1% (v/v) NGS and 0.2% (w/v) saponin in PBS; primary antibodies and concentrations are listed in Fluorescent images of brain slices were obtained using an IX83 inverted-imaging system with a DP80 camera and 10x, 20x, or 40x UPlanSApo (0.75 NA) and 60x UPlanSApo oil-immersion (1.35 NA) objectives .RRID:SCR_013673), cellSens Dimension version 1.18 (Olympus) or Fiji . Image analysis of protrusions was performed using cellSens Software (Olympus).For confocal-image acquisition, either an upright TCS SP8 Laser microscope equipped with a 63x objective  using sequential scanning with the 488 nm line of an argon-ion laser and the 543 nm line from helium–neon lasers  (Leica Microsystems) or an inverted confocal laser scanning FV3000 microscope equipped with 10x (0.75 NA), 20x (0.8 NA), and 40x  objectives using sequential scanning with 405 nm, 488 nm, 561 nm cw diode lasers  (Olympus) was used. Background correction and adjustment of brightness and contrast were performed using either LasX confocal software version 3.5.7 . After centrifugation, the topmost layer of 0.32 M sucrose contained the cytosolic fraction, the band between 0.32 and 0.85 M sucrose contained the myelin-enriched fraction, the band between 0.85 and 1 M sucrose contained the Golgi- and ER-enriched fraction, the band between 1 and 1.2 M sucrose contained the synaptosomal fraction, and the pellet consisted of the mitochondria-enriched fraction. The synaptosomal fraction was further centrifuged at 33,000 × g at 4°C for 20 min and the resulting pellet was re-suspended in 6 mM Tris/HCl pH 8.0 for hypo-osmotic shock. The solution was again centrifuged at 33,000 × g at 4°C for 20 min. The resulting supernatant contained synaptic cytoplasm and the crude synaptic-vesicle fraction. The crude synaptic-plasma membrane fraction was extracted by re-suspending the pellet with 6 mM Tris/HCl pH 8 and 1% (v/v) Triton X-100. The crude synaptic plasma membrane fraction was then centrifuged a 33,000 × g at 4°C for 10 min; the resulting pellet contained the postsynaptic density fraction.Subcellular fractionation was adapted from previously described protocols . Adult m2) prior to washing, reduction, alkylation and tryptic digestion ; 1 missed cleavage site allowed; oxidation M variable modification; carbamidomethyl C fixed modification. A summary of identified proteins is provided in Following electrophoresis, gels were stained with Coomassie Brilliant Blue  and slicigestion . The resigestion . Elutingp ≤ 0.05 was adopted . Values were analyzed for normal distribution using the D’Agostino and Pearson test. Statistical analysis was performed using the 1 way ANOVA . All data are presented as mean + SEM. A level of confidence of The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern (LALLF) and Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES).AB, IG, and NB designed the study. AB and IG wrote the manuscript, with contribution from all co-authors. IG performed western blot experiments and immunostainings and analyzed the results. DV performed primary neuron transfection and immunostainings. FK performed western blot experiments. NB performed and analyzed activity inhibitor experiments. LW and RR performed the proteomic analysis. MW helped with RMS analysis. AH, TP, and MH revised the manuscript. Data from this work was part of the Ph.D. theses of IG, DV, and FK. All authors read and approved the final manuscript.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.


Phagocytic activity of both neutrophils and monocytes were significantly decreased in sepsis (p = 0.002 and p = 0.0003). Sepsis plasma stimulated healthy neutrophils, showed significant increase in NETs (neutrophil extracellular traps) and cell free DNA (p = 0.049 and p = 0.04) compared to w/o sepsis and HC. Proteomic analysis revealed upregulated- DNAJC13, TMSB4X, GPI, GSTP1, PNP, ANPEP, COTL1, GCA, APOA1 and PGAM1 while downregulated- AHSG, DEFA1,SERPINA3, MPO, MMRN1and PROS1 proteins  associated to neutrophil activation and autophagy in sepsis. Proteins such as DNAJC13, GPI, GSTP1, PNP, ANPEP, COTL1, PGAM1, PROS1, MPO, SERPINA3 and MMRN1 showed positive correlation with neutrophils activity and number, oxidative burst activity and clinical parameters such as MELD, MELD Na and Bilirubin. Proteomic analysis revealed that faulty functionality of neutrophils may be due to the autophagy proteins i.e., DNAJC13, AHSG, TMSB4X, PROS1 and SERPINA3, which can be used as therapeutic targets in decompensated cirrhosis patients with sepsis.Decompensated cirrhosis (DC) is susceptible to infections and sepsis. Neutrophils and monocytes provide the first line of defense to encounter infection. We aimed to evaluate proteins related to neutrophils functionality in sepsis. 70 (DC), 40 with sepsis, 30 without ( Liver cirrhosis is the end stage of chronic liver disease due to viral (hepatitis B and hepatitis C) and non-viral etiologies  ,2. NeutrIn cirrhosis, translocation of gut bacteria to the liver is enhanced due to increased membrane permeability, causing endotoxemia and systemic inflammation with dysregulated immune cell activation. Compromised immunity of cirrhotic patients made them more prone to multiple infections, including spontaneous bacterial peritonitis (SBP), urinary tract infection, pneumonia, cellulitis resultant sepsis and septic shock with multiple organ dysfunction in 20–40% patients ,8,9.Chemokine receptors CXCR1 and CXCR2 drive neutrophil migration to the inflammation site in response to its ligand CXCL8/IL-8, leading to the hepatic inflammatory response , althougDuring sepsis, autophagy uses a cellular adaptive defensive method for limiting cell damage and apoptosis, as well as removing bacteria and pathogens. The autophagy process even showed altered effects on the different stages of neutrophil differentiation. Any deficiency in autophagy in neutrophils significantly reduced ROS generation, NADPH oxidase, and degranulation in vitro and in vivo. Both autophagy and ROS generation are mainly required for chromatin decondensation in NETosis, and impaired autophagy causes a decrease in NETosis, leading to the survival of neutrophils which directly increase the mortality in sepsis patients ,19,20.Our aim was to identify the unique proteins related to neutrophils functionality in decompensated cirrhosis with sepsis.n = 30), with sepsis (n = 40) and healthy control  at Institute of Liver and Biliary Sciences (ILBS), New Delhi between 2017 and 2019. This study was approved by the Research and Institutional ethics committee of ILBS with IEC No IEC/2016/45/NA/C2, and informed consent was obtained from all the subjects enrolled in the study. In this longitudinal study, all the patients were closely monitored from the time of admission and studied at baseline, day 3 and 7. This study was carried out in accordance with the ethical standards of the Helsinki declaration.We enrolled 70 decompensated cirrhosis patients: without Sepsis , with HCC or any other site malignancy or any other co-morbidities, and no consent given were excluded from the study. The clinical as well as biochemical assessment of all the enrolled patients with or without SIRS (systemic inflammatory response syndrome) or sepsis patients was done according to the treating physician’s direction. Patients were recruited based on the following criteria:w/o) sepsis was diagnosed when there was no evidence of SIRS, or infection on any of the cultures and ultrasonography.Decompensated cirrhosis without  score with an infection, confirmed either by cultures or by imaging.(a)Temperature >38.0 °C or <36.0 °C(b)9/LTLC  < 4 or >12 × 10(c)Pulse > 90 beats/minutes(d)RR (Respiratory rate) >20 or <32 beats/minutesFour criteria for the diagnosis of SIRS were used :(a)TempeAccording to the institutional protocol, medications were given to:DC patients without sepsis: For standard indications , IV fluids and albumin are given however prophylactics and third generation cephalosporin antibiotics were given to the patients admitted in ICU/HDU.DC patients with Sepsis: Broad-spectrum antibiotics  were given to the patients according to the site , severity (sepsis with organ dysfunction or septic shock), and acquisition of the infection .10–15 mL of peripheral blood was collected at room temperature in an ethylene diamine tetraacetic acid (EDTA) coated and heparin tubes from patients and healthy subjects at the time of recruitment in the study. Peripheral blood was used for whole blood immunophenotyping, phagocytic activity, oxidative burst activity, expression of neutrophilic CD64 and monocytic HLA-DR (nCD64-mHLADR Index), and neutrophils isolation. From the rest blood, plasma was separated for assessment of cytokines, endotoxin, and other circulating proteins, whereas plasma devoid blood was used for isolation peripheral blood mononuclear cells (PBMCs).Endotoxin levels were estimated using PierceTM Chromogenic Endotoxin Quant Kit . All reagents and standards were prepared as per the manufacturer protocol freshly before use. Plate was pre-equilibrated at 37 ± 1 °C heating block and maintained for whole procedure time. 50 µL of blanks, standards, and samples added/well followed by addition of 50 µL of the reconstituted amebocyte lysate reagent/well. After addition of amebocyte lysate reagent, the plate was removed from the heater and mixed gently by tapping 10 times on the sides of the plate and returned to the plate heater at 37 ± 1 °C. Chromogenic substrate was reconstituted with endotoxin free water and pre-warm at 37 ± 1 °C for 5 min before use. 100 µL of chromogenic substrate was added and mixed gently by tapping 10 times on the sides of the plate. 50 µL of stop solution (25% acetic acid) was added/well while keeping the plate at heater at 37 ± 1 °C and mixed gently by tapping 10 times. Optical density (OD) of the plate was immediately taken at 405 nm using Multiskan EX . Average absorbance of blank to be subtracted from all standards and samples replicates for calculation of mean ∆ absorbance. Standard curve was prepared using blank subtracted absorbances for the standards with endotoxin concentration in EU/mL. Linear regression with the coefficient of determination was used to determine the endotoxin concentration of each sample.Neutrophils and monocytes were characterized in whole blood using specific antibodies against surface markers labelled with different fluorochromes. Neutrophils were characterized using anti-CD11b at 1:100 dilution , anti-CD66b at 1:40 dilution , anti-CXCR2 at 1:40 dilution , and anti-CXCR1 at 1:40 dilution . Monocytes were characterized using anti-CD14 at 1:40 dilution , anti-CD16 at 1:30 dilution , and anti-HLA-DR at 1:40 dilution  .Briefly, 100 µL of whole blood was incubated with appropriate concentrations of surface antibodies for 30 min at room temperature in the dark. After that, freshly prepared 1× RBC lysis solution added in the cells and incubated for 5 min at room temperature, dark and washed with 1× PBS. A minimum of 100,000 events were acquired using BD FACS VERSE, and all relevant data was analyzed by using FlowJo software (version 10).E. coli bacteria, quenching solution, lysis solution and wash solution provided in the ready to use phagotest kit from Celonic, Switzerland #341060. 100 µL of heparin blood was used and mixed with 20 µL precooled E. coli bacteria (4 × 107 bacteria). Both test sample and control tube were incubated in water bath for 10 min at 37 °C. After 10 min, tubes were kept on ice to stop phagocytosis. 100 µL of ice-cold quenching solution added to each tube and mixed properly using vortex mixer. 3 mL of wash solution added twice to the tubes and centrifuged for 5 min, 250× g, 2–8 °C and supernatant was discarded. After that, pre-warmed (room temperature) 1× lysis solution was added to the samples, vortexed and incubated for 20 min at room temperature, then centrifuged for 5 min, 250× g, 2–8 °C and supernatant was discarded. Again, 3 mL of wash solution added to the tubes and centrifuged for 5 min, 250× g, 2–8 °C. After washing twice, samples were ready to be acquired within 60 min. 100,000 events/tube acquired in BD FACS VERSE. Neutrophils and monocytes were gated to analyze the phagocytic activity using FlowJo (version 10).Phagocytic activity was performed using manufacturer protocol . All the reagents i.e., opsonized FITC labelled E. coli bacteria (particulate stimulus), FMLP , PMA (high stimulus), dihydrorhodamine 123, lysis solution and wash solution provided in the ready to use oxidative burst kit from Celonic, Switzerland #341058. Accordingly, 100 µL of heparin blood was incubated with 20 µL of each with final concentration; precooled E. coli bacteria (2 × 107 bacteria), FMLP (5 µM), and PMA (8.1 µM), and incubated for 10 min at 37 °C in pre-warmed water bath. Both FMLP and PMA reagents were freshly prepared in wash solution immediately before the experiment. Oxidative burst activity was performed using manufacturer protocol . All the reagents i.e., opsonized g at 2–8 °C, and supernatant was discarded. After washing twice, samples were ready to be acquired within 30 min. 100,000 events/tube were acquired in BD FACS VERSE. Neutrophils and monocytes were gated to analyze the oxidative burst activity using FlowJo (version 10).After incubation, 20 µL of dihydrorhodamine 123 (substrate solution) was added in all tubes, except unstained and incubated for 10 min at 37 °C in water bath, followed by addition of the 1ml of pre warmed 1× lysis solution. Tubes were vortexed and incubated further for 20 min at room temperature. Again, 3 mL of wash solution added to the tubes, centrifuged for 5 min at 250× The expression of nCD64 and mHLA-DR was used for calculating nCD64-mHLADRindex by using BD Quantibrite™ CD64/CD45  and BD Quantibrite™ Anti-HLA-DR/Anti-Monocyte , as per the protocol. Briefly, for staining, 50 μL of whole blood was mixed with 20 μL of conjugated monoclonal antibody and incubated for 60 min in the dark at 20–25 °C, room temperature. After incubation, 2 mL of 1× BD FACS lysing solution  was added and further incubated for 30 min. Finally, cells were washed and quantitation acquisition was done in BD FACS Calibur using BD CellQuest™ software.= Geometric mean of CD64 neutrophils.nCD64-mHLADR index was calculated following this formula:= Geometric mean of HLA-DR monocytes.mHLA-DR g for 30–35 min at room temperature. After centrifugation, two leucocytes’ bands were visible. The top band at the sample interface consisted of mononuclear cells and the lower band of neutrophils. The lower band of neutrophils harvested into a new centrifuge tube with pasteur pipette. Cells were washed with 1× PBS and centrifuged at 400× g for 10 min. Supernatant removed and resuspended the cells in 1 mL of PBS. Cells counted using hemocytometer. Purity of the cell preparation can be determined by flow cytometry and viability by trypan blue exclusion. Isolated neutrophil cells were further used for NETosis.Neutrophils were isolated using polymorphoprep  at room temperature immediately after collection of peripheral blood. Anti-coagulated whole blood was layered on top of polymorphoprep layer in 1:1 ratio in a 15 mL centrifuge tube and centrifuged at 500× 2 incubator overnight. After that, the plate was removed from CO2 incubator, washed with autoclaved distilled water, and dried. 5 × 105 isolated neutrophil cells plated/well in RPMI media  with 10% FBS  and 1% penicillin-streptomycin  and incubated in CO2 incubator for 2 h for the attachment of neutrophils. After 2 h, all media were removed and replaced with fresh RPMI media along with stimulations i.e., PMA , LPS , rIL-8  and PLASMA (1:10 dilution). After that, the plate was incubated again in CO2 incubator for 4–6 h. After that, media was removed from all wells and collected for estimation of cell free DNA, cells fixed with 4% paraformaldehyde for 10–15 min. After 15 min, cells were washed with 1× PBS and coverslips mounted on slides with DAPI-mount . Slides were used to take images of NETs using a Nikon Intense light Microscope.Round coverslips were coated with 1x poly-lysine , placed in 12 well plate and incubated in COFor cell free DNA estimation, media removed after 4–6 h of stimulation was used. Salmon sperm DNA  was used as standards at different concentrations. Samples and standards were stained with sytox green  for 5 min and plated in duplicates in black cell culture plates . Fluorescence of sytox green was detected at 485/520 (Exi/Emi) using FLUOstar Optima .To understand the significance of various cytokines and growth factors associated with sepsis and neutrophils, we analyzed forty-one cytokines, chemokines, and growth factors such as IL-1RA, IL-1β, IL-2, IL-12 (p40), IL-27, IL-18, IL-10, IL-17A, IFN-γ, TNF-α, IL-4, IL-6, IL-33, TGF-β1, IL-8, MIF, MIP-1α, MIP-1β, MIP-3α, ITAC, MCP-1, FRACTALKINE, ENA78, IP-10, EOTAXIN, Angiopoientin, MCSF, G-CSF/CSF3, GM-CSF, HGF, LEPTIN, TPO, VEGF-A, MMP7, MMP8, MMP1, MMP12, MMP13, P SELECTIN, E SELECTIN and TREM1 in plasma by using custom multiplex PROCATAPLEX , following the complete details on Luminex xponent 3.1TM Rev.2 . Standard curve was drawn using standards provided in the kit. Values in samples were determined corresponding to the standard curve drawn. The lower limits of detection of the test for each analyte is provided in the In brief, plasma samples used for proteomic analysis were diluted with 0.1% formic acid in 1:5 ratio and run through an AKTA pure protein purification system (Cytiva) using UNICORN 7.1 software in order to deplete albumin. Albumin depleted supernatant was collected and used for protein estimation using Bradford reagent . A total of 30 ug of proteins were dissolved in 50 mM ammonium bicarbonate and reduced by dithiothreitol (DTT) for 40 min at 56 °C. Proteins were then alkylated by 20 mM iodoacetamide (IAA) for 40 min at room temperature in the dark. The 30 μg of protein samples were then kept for digestion with 1 μg sequencing grade trypsin  at 37 °C. The reaction was stopped by acidifying the peptides using 0.1% formic acid followed by desalted on reversed-phase C-18 spin columns . The peptides were then eluted using 50 µL of 60% acetonitrile in 0.1% formic acid and concentrated in a speed vac machine. The peptides were finally acidified with 2% acetonitrile, 0.1% trifluoroacetic acid in 0.1% formic acid. These peptides were subjected to nano-electrospray ionization and Tandem mass spectrometry (MS/MS) using Q-ExactiveTM Plus  at the collision-induced dissociation mode with the electrospray voltage was 2.3 kV. Further data was analyzed using proteome discoverer . Uniprot Homo sapiens (Human) database (UP000005640) with Mascot algorithm  was used. Identified proteins were subjected to standard statistical analysis and network and pathway analysis .Total RNA was isolated from neutrophils using miRVANA kit . cDNA was prepared from RNA using High-Capacity cDNA Kit . mRNA expression of genes like DNAJC13, TMSB4X, AHSG, ATG5, ATG12, HMGB1, and HIF-1α were analyzed. The primer sequences of the genes are listed in p-values <0.05 were considered statistically significant.Data were analyzed using the statistical software Prism  and SPSS version 22 . The comparison for continuous data is carried by using One-way ANOVA/Kruskal–Wallis test followed by probability adjustment by the Mann–Whitney test or by Bonferroni test post-hoc comparison as appropriate and it is represented as mean ± standard deviation (SD). The w/o sepsis  and 40 patients with sepsis  and 15 age-matched healthy controls were analyzed at the time of admission and enrolment in the study. Alcohol was the predominant etiology (70%) in DC patients. Sepsis patients showed a significant increase in total bilirubin, AST levels, INR, PCT, lactate, MELD Na, SOFA score, creatinine, TLC, and pulse compared to w/o sepsis in Baseline characteristics of 70 DC patients, 30 patients w/o sepsis and HC (p = 0.028 and p = 0.042) (Plasma endotoxin levels were found to have increased in the sepsis patients compared to = 0.042) A.p = 0.0076, p = 0.008) compared to w/o sepsis. No significant difference was observed in the percentage of monocytes between the groups  showed an increase in the percentage of neutrophils and neutrophil-to-monocyte ratio (NMR) in sepsis patients (e groups B.w/o sepsis patients compared to HC (p < 0.0001 and p = 0.004), although it was two-fold more increased in sepsis patients compared to w/o sepsis, but no significant difference was observed between sepsis and w/o sepsis   D,E and ss and HC E. It clew/o and with sepsis patients. However, percentage frequencies of CD11b+CD66b+ and CXCR2+ on CD11b+CD66b+ were decreased in sepsis patients at day 7 (p = 0.005 and p = 0.047) compared to day 0. However, percentage frequencies of CD11b+CD66b+, CXCR1+ and CXCR2+ on CD11b+CD66b+ were increased in w/o sepsis patients at day 7  compared to day 0  and other etiology (NASH and cryptogenic), we have found no difference in the percentage of neutrophils CD11bh sepsis . Consideh sepsis .+CD66b+ neutrophils, CXCR1+, and CXCR2+ on CD11b+CD66b+ cells in sepsis patients   A,B.E. coli (particulate stimulus). Spontaneous (SPT) and FMLP stimulated oxidative burst activity of neutrophils showed no difference between the groups. PMA, being a strong stimulant, still showed decreased oxidative burst activity of neutrophils in sepsis compared to w/o sepsis and HC. However, E. coli stimulated neutrophils showed a significant increase in oxidative burst compared to spontaneous in all the groups, i.e., HC (p < 0.0001), w/o sepsis (p < 0.0001) and sepsis (p < 0.0001), but E. coli stimulated neutrophils had decreased oxidative burst activity of neutrophils in sepsis patients (p = 0.028) compared to HC , PMA (high stimulus), and ed to HC C,D. ThisNo significant difference in phagocytic and oxidative burst activity of neutrophils was observed in different time points between the groups .w/o sepsis and HC plasma. As positive controls, rIL-8 stimulated neutrophils showed many NETs and LPS stimulated neutrophils showed very few NETs (w/o sepsis (p = 0.049) and HC plasma (p = 0.04). Though w/o sepsis patient plasma also showed a significant increase in cell-free DNA compared to HC (p = 0.044)  F.No significant difference in cell free DNA concentration was observed at different time points between the groups .p = 0.045) compared to HC, but no significant difference was observed between sepsis and w/o sepsis patients (p = 0.007) compared to w/o sepsis. Whereas classical and intermediate monocytes showed no difference in the groups (w/o sepsis and HC (p = 0.042 and p = 0.03) but no difference in expression of HLA-DR on classical and intermediate monocytes (Expression of HLA-DR on monocytes (mHLA-DR) in the peripheral blood was significantly reduced in sepsis patients  compared to day 0. There was no difference in the expression of HLA-DR at follow-up time points in classical and intermediate monocytes, but HLA-DR expression on non-classical monocytes was found significantly increased in w/o sepsis patients at day 7 (p = 0.01) compared to day 0  compared to HC, but no significant difference was observed between sepsis and w/o sepsis . However, E. coli stimulated monocytes had increased oxidative burst activity compared to spontaneous activity in HC (p = 0.007), w/o sepsis (p = 0.002) and sepsis (p = 0.03) (Spontaneous (SPT) and FMLP stimulated monocytes displayed no difference in oxidative burst activity in all groups. PMA stimulated monocytes also showed a significant decrease in oxidative burst activity in sepsis compared to HC ( = 0.03) C,D.No significant difference in both the phagocytic and oxidative burst activity of monocytes was observed at different time points between the groups .Plasma samples were analyzed for soluble factors using proteomics, as well as cytokines, chemokines, and growth factors using cytokine bead array A.w/o sepsis and HC, and 26 in sepsis and w/o sepsis, while 12 downregulated proteins were uniquely expressed in sepsis and HC, 32 in sepsis and HC, and 24 in sepsis and w/o sepsis  revealed a clear separation of plasma proteins in three different groups B. Furtheo sepsis C.w/o sepsis patients were GPI, DNAJC13, and APOA1 ) (Gene ontology (GO) annotation analysis explained these uniquely expressed proteins, in their biological significance, cellular localization, and molecular functions. It was observed that upregulated proteins in sepsis patients compared to HC were DNAJC13, GPI, GSTP1, PNP, ANPEP, COTL1, GCA, PGAM1, and TMSB4X and compared to 034774)) D.w/o sepsis, patients were AHSG, PROS1, DEFA1, SERPINA3, MPO, and MMRN1 ) ) D. Out of034774)) E.w/o sepsis and HC  of upregulated proteins shows decreased at day 7 compared to day 0 A. While w/o sepsis patients at follow-up of day 3 and day 7 time points, cytokines, i.e., IL-6, IL-8, IL-18, IL-33, MIF, MIP1a, MIP1b, and MIP3a were increased, and IP-10, MCSF, and ESelectin were decreased compared to day 0  compared to HC and DNAJC13 and AHSG were found to decrease in sepsis compared to w/o sepsis , though another autophagy gene ATG5 was increased but not significant in sepsis. Further HIF-1α expression (p = 0.046) was found to decrease in sepsis; however, HMGB1 was decreased but not significant in sepsis compared to w/o sepsis patients  is mainly present on the surface of neutrophils and gets upregulated by proinflammatory agonists, i.e., fMLF, IL-8, and TNF-α. The use of proinflammatory agonist TNF-α enhances the neutrophils apoptosis, which gets regulated by ANPEP in humans, and which can be reversed by the ANPEP inhibitors such as actinonin or bestatin [2+-binding protein, i.e., GCA  mainly expressed in neutrophils and monocytes or macrophages. GCA-deficient neutrophils cause the recruitment to the inflamed site and activation of neutrophils [Out of these upregulated and downregulated proteins, many proteins were known to play an important role in the innate immune system and neutrophils processes. ANPEP  member C13) protein acts as a positive modulator of autophagy and maintains cellular homeostasis by ATG9A trafficking . AHSG (AWe conclude that the faulty functionality of neutrophils may be due to the autophagy proteins, i.e., DNAJC13, AHSG, TMSB4X, PROS1, and SERPINA3, which can be used as therapeutic targets in decompensated cirrhosis patients with sepsis.