H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde,whichwas synthesized by copper(I)-catalyzed azide–alkyne cycloaddition(CuAAC). The structure of the compound was analyzed using Fouriertransform infrared spectroscopy (FTIR), 1H NMR, 13C NMR, UV–vis, and elemental analyses. Moreover, X-ray crystallographystudies demonstrated that the compound adapted a monoclinic crystalsystem with the P21/c space group. The dominant interactions formed in the crystal packingwere found to be hydrogen bonding and van der Waals interactions accordingto Hirshfeld surface (HS) analysis. The volume of the crystal voidsand the percentage of free spaces in the unit cell were calculatedas 152.10 Å3 and 9.80%, respectively. The evaluationof energy frameworks showed that stabilization of the compound wasdominated by dispersion energy contributions. Both in vitro and insilico investigations on the DNA/bovine serum albumin (BSA) bindingactivity of the compound showed that the CT-DNA binding activity ofthe compound was mediated via intercalation and BSA binding activitywas mediated via both polar and hydrophobic interactions. The anticanceractivity of the compound was also tested by the 3--2,5-diphenyltetrazoliumbromide (MTT) assay using human cell lines including MDA-MB-231, LNCaP,Caco-2, and HEK-293. The compound exhibited more cytotoxic activitythan cisplatin and etoposide on Caco-2 cancer cell lines with an IC50 value of 16.63 ± 0.27 μM after 48 h. AnnexinV suggests the induction of cell death by apoptosis. Compound 3 significantly increased the loss of mitochondrial membranepotential (MMP) levels in Caco-2 cells, and the reactive oxygen species(ROS) assay proved that compound 3 could induce apoptosisby ROS generation.We report herein a new 1,2,3-triazole derivative, namely,4--1 Thesesignificant biological and medicinal properties of triazoles haveencouraged investigations into the synthesis and detailed structuralcharacterization of new 1,2,3-triazole derivatives.7 TheCu(I)-catalyzed azide–alkyne cycloaddition, also known as theCuAAC “click reaction,” significantly improved the preparationof 1,4-disubstituted 1,2,3-triazole-based compounds under mild conditions.9Cancer is one of the mostcommon diseases in the world, causingthe deaths of millions of people every year.12 A few triazole compoundshave also been transferred to clinical trials. Among them, tazobactam,which is a non-nucleoside inhibitor of reverse transcriptase activity,13 β-lactamase,14 carboxyamidotriazole (CAI), which are signal transduction blockers,15 letrozole, and anastrozole, which are triazolederivative drugs targeting aromatase, are used to treat breast cancer.15Especially, 1,2,3-triazole derivatives have been thoroughlystudiedas potent tools in anticancer research.18 Anticancer drugs bind to DNA mainly in three modes: electrostaticattraction, groove binding, and intercalation.19 However, anticancer drugs interact with DNA in a varietyof complex ways, the exact mechanisms of which are still unknown andneed to be deeply investigated. In these circumstances, research intohow compounds interact with DNA is critical for the design and developmentof anticancer drugs that are effective against a wide range of diseases.An effective interaction between a smalldrug molecule and DNAor proteins typically dictates its anticancer activity. For this reason,it is critical to examine binding interactions between DNA and anticancercompounds in order to find effective anticancer drugs.20 The investigation of the drug–proteininteractions is also critical, since most of the drugs, which arebound to serum albumin, are transported as drug–protein adducts.21In addition to DNA, proteins have also attracted the attentionof researchers as prime molecular targets. Albumins are the most importantproteins found in blood plasma, transporting both endogenous and exogenouscompounds.24Molecular docking is one of the most populartechniques used forexamining the molecular mechanisms of interactions formed betweensmall compounds and their potential biomacromoleculer targets includingDNA or bovine serum albumin. The use of molecular docking to identifypreferred sites for binding and the optimum orientation of drug candidateson a target protein can be helpful when designing chosen analogueswith higher activities, as this information reveals the binding affinityand potential activity of the drug candidate.Despitethe market’s abundance of anticancer medicationsand the fact that many are currently undergoing clinical trials, thereis an urgent need for the development of effective, targeted, lesstoxic anticancer drugs to treat the millions of new cases of cancerdiagnosed each year.H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde as a new 1,4-disubstituted1,2,3-triazole derivative. The compound was characterized using elementalanalyses, Fourier transform infrared spectroscopy (FTIR), 1H and 13C APT NMR, and single-crystal X-ray diffraction(SCXRD). The HS analysis, interaction energy and energy frameworkinvestigations, and the volume of the crystal voids of the compoundwere also performed. Additionally, DNA and bovine serum albumin interactionproperties of the synthesized compound were further studied by applyingin vitro and in silico methods. Finally, its anticancer activity wasevaluated using a 3--2,5-diphenyltetrazoliumbromide (MTT) assay against cancer cell lines including MDA-MB-231(breast cancer), LNCaP (prostate carcinoma), Caco-2 ,and HEK-293 . Annexin V, mitochondrial membranepotential (MMP), and reactive oxygen species (ROS) assays were alsocarried out.Therefore, in light of these facts, wehave decided to design anovel molecule that can be used as an anticancer agent. We used clickchemistry to synthesize 4--122.1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde (3) was synthesized by the copper(I)-catalyzed azide–alkynecycloaddition (CuAAC) reaction between 2-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde(1) and 4-azido-1,2-dichlorobenzene (2)with the addition of CuAc as a catalyst and NaAsc as a reducing agentusing dichloromethane/water (1:1) as a solvent system -1t system 1.Figure S5,the absence of an azide peak belongs to 4-azido-1,2-dichlorobenzeneat 2120 cm–1 and the presence of the peak at 3155cm–1 belongs to the target compound, showing theformation of the 1,2,3-triazole ring.25 In the IR spectra of compound 3, the ν(C=O)stretching vibrations of benzaldehyde carbonyl were determined at1644 cm–1 and ν(C–O) is identifiedat 1218 cm–1.26In the IR spectraof the compound, as shown in 1H NMR spectrum of 4--1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde showeda singlet peak at δ 8.08 ppm due to the −CH of the 1,2,3-triazolering, which confirms the formation of the 1,2,3-triazole moiety (2−),respectively. The aromatic protons appear as doublets at the δvalues of 6.56, 6.64, 7.47, 7.62, and 7.91 ppm.28The e moiety 1. Single13C NMR was applied as attachedproton test (APT),where the signals belong to the CH/CH3 yield positive andsignals belong to the C/CH2 yield negative. 13C NMR spectra of the compound showed peaks at δ values of 119.66and 144.38 ppm, which were attributed to the C-5 and C-4 carbon atomsof the 1,2,3-triazole ring, respectively. The observed peak at 62.19ppm was assigned to the methylene carbon attached to the C-4 positionof the triazole ring. The signals belonging to the aldehyde carbonylcarbon atom appeared downfield at a δ value of 194.69 ppm. Thesignals at δ values of 165.15, 164.49, 135.93, 135.68, 134.27,133.40, 131.72, 122.50, 121.10, 115.82, 108.56, and 101.92 ppm wereassigned to the carbon atoms of aromatic rings. 13C NMRsignal directions were in good agreement with the type of carbon atomsand had a good correlation with 1H NMR spectra when consideringthe attached protons to each carbon atom.The 31 The molecularformula was confirmed by 1H NMR, 13C APT NMR,FTIR, UV–vis, and elemental analysis.Spectral characterization data were similar tothose previouslyreported for 1,2,3-triazole derivatives.2.23 wasconfirmed using single-crystal X-ray structural analysis. Thespectroscopic data-based structure assignment of A (C1–C6), B (N1–N3/C7/C8),and C (C10–C15), respectively. So, they arealmost coplanar with adjacent rings.The asymmetric unit along with the atom numberingscheme is depictedin A/B = 35.76(8)°, A/C = 47.00(7)°, and B/C = 12.37(8)°. There is an intramolecularO–H···Ohydrogen bond  ring motifs.32 Further, π–π interactionsbetween planar, C (C10–C15) rings, and [Cg3···Cg3i] of neighboring molecules help to consolidatethe crystal packing .In the crystal structure of tructure 3, enclos2.33 analysis was carried out in order to visualize the intermolecularinteractions in the crystal of 3,34 by user dnorm 4 denotes38 as shown in In addition to appearing as blue and red regionscorrespondingto positive and negative potentials, respectively, on the HS mappedover electrostatic potential3.Hydrogen-bond donors are represented by blue regions,which havepositive electrostatic potential, while hydrogen-bond acceptors arerepresented by red regions. The shape index of the HS is a tool tovisualize the π···π stacking by the presenceof adjacent red and blue triangles; if there are no adjacent red and/orblue triangles, no π···π interactions arepresent. de and id combinations across the surface of the molecule.The color of each point corresponding to the relative area of each de and id combinationis recognized as the contribution from different interatomic contacts.The uncolored region denotes no contribution to the HS, while theblue, green, and red correspond to the smallest, moderate, and largestcontributions, respectively. The overall 2D fingerprint plot, 39 are illustrated in de + id = 2.86 Å.The H···H contacts are reflected in de = id = 1.24 Å. In the absence of C–H···πinteractions, the pair of characteristic wings in the fingerprintplot delineated into H···C/C···H contacts fingerprint plots, which show the visual contentsof the frequencies of the ·H9.9%, 7e andH··H9.9%, 7f contac·C 7.0%, 7g contac·C5.2%, 7h andO··C5.2%, 7i contac·C 3.6%, 7j, C···C·C 3.6%, 7k,N···C·C 3.6%, 7l, O···O·C 3.6%, 7m, and N·C 3.6%, 7n contacdnorm plotted onto the surface are shown forthe H···Cl/Cl···H, H···H,H···C/C···H, and H···O/O···Hinteractions in The nearest neighbor coordination environment ofa molecule canbe determined from the color patches on the HS based on how closeto other molecules they are. The HS representations with the function 40The HS analysis confirms the significance of H-atomcontacts inpacking formation. The abundance of H···Cl/Cl···H,H···H, H···C/C···H, andH···O/O···H interactions suggests thatvan der Waals interactions and hydrogen bonding play major roles inthe crystal packing.2.441 The void surface is calculated for the entireunit cell and defined as an isosurface of the procrystal electrondensity, where the void surface meets the boundary, and the cappingfaces are generated to form an enclosed volume. The volume of thecrystal voids  is the sum of electrostatic (Eele), polarization (Epol), dispersion(Edis), and exchange–repulsion(Erep) energies43 with scale factors of 1.057, 0.740, 0.871, and 0.618, respectively.44 Hydrogen-bonding interaction energies (in kJmol–1) are −18.7 (Eele), −4.2 (Epol), −24.2(Edis), 23.7 (Erep), and −29.3 (Etot) for C2–H2···N2; −10.2 (Eele), −1.1 (Epol), −43.4(Edis), 25.5 (Erep), and −33.6 (Etot) for C6–H6···N3; and −15.3 (Eele), −2.9 (Epol),−73.3 (Edis), 44.2 (Erep), and −54.8 (Etot) for C14–H14···O2.The intermolecular interaction energies are calculatedwith the CE–B3LYP/6-31G energy model available in CrystalExplorer 17.5,43 Energies between molecular pairs are representedby cylinders joining the centroids of two molecules, with the radiusof the cylinder proportional to the relative strength of the correspondinginteraction energies, which were scaled to the same factor of 80 witha cutoff value of 5 within 2 × 2 × 2 unit cells. Eele (red cylinders), Edis (green cylinders), and Etot (blue cylinders). The evaluation of the electrostatic, dispersion,and total energy frameworks shows that the dispersion energy contributiondominates the stabilization.Thecalculation of intermolecular interaction energies is combined witha graphical representation of their magnitude in energy frameworks.2.645 For this reason, the interactions between compound 3 and CT-DNA were investigated by measuring the absorption spectralchanges when CT-DNA was added to a fixed concentration of the compound. 3 in the absence and presence ofincreasing concentrations (0–100 μM) of CT-DNA alongwith the inset of the Wolfe–Shimmer plot. Kb).The use of absorptionspectral titration to determine the interactions of potent anticanceragents with CT-DNA is an effective method.3 showedan absorptionband at 272 nm, and this band revealed hypochromicity (86.18%) withincreasing concentrations of CT-DNA. Upon the addition of CT-DNA,the absorption spectrum of compound 3 also showed a bathochromicshift (Δλ = 13 nm) within the bands at 272–285nm, indicating an interaction between the electronic states of chromophoresand DNA bases, possibly as a result of the intercalation. These observationsare common for compound–DNA non-covalent interactions.46 Additionally, the isosbestic point at 370–390nm indicates an equilibrium between the free and CT-DNA bound compound.47The UV–vis spectra of Kb)was calculated based on titration data using the Wolfe–Shimmerequation.48Kb was determined by the slope-to-y-intercept ratioin the plot of [DNA]/(εa – εf) versus [DNA], as detailed in the SI.The intrinsic binding constant .57 In the case of intercalation, general spectral absorption changesfor intercalation have been expected to be bathochromic (>15 nm)andhypochromic (>35%), whereas groove binding results in no bathochromismor around 6–8 nm shift.58 In lightof these facts, our findings suggest that compound 3 wasbound to CT-DNA via intercalation. Furthermore, ΔG for the DNA–compound 3 interaction was foundto be −31.25 kJ·mol–1. A negative valuerevealed the spontaneity of the binding. However, further experimentswere definitely needed to confirm the intercalation binding mode becauseof the lower bathochromic shift than typical intercalation interactions.The 59 If compound 3 orany compound competes with EB and intercalated between base pairsof EB-bound DNA, the emission will be decreased after the displacementof the EB molecules from the CT-DNA.EB displacement studies were also employed using fluorescence spectroscopy.EB is a well-known DNA intercalator, intercalating with CT-DNA throughits planar phenanthroline moiety to form an EB + CT-DNA complex. EBshows weak fluorescence intensity when excited around 520 nm. Intercalationof EB into DNA base pairs significantly increases the emission intensityof EB.3 (0–150 μM)were added into constant concentrations of [EB (5 μM) + CT-DNA(25 μM)] solution while the spectral changes in the fluorescenceemission were monitored at 602 nm after excitation at λex 510 nm. With the addition of increasing concentrations ofcompound 3 into the solution containing a fixed concentrationof the EB + CT-DNA adduct, a decrease in the emission intensity ofthe 602 nm band is observed, as shown in 3 indicated that thestudied compound displaced the EB from CT-DNA, which is a characteristicof the intercalative binding mode.60Increasing amounts of compound Ksv) was calculated as 4.13 × 103 M–1 from the slope of the plot of I0/I versus [Q] givenin 3 according to the Stern–Volmerequation (Io/I = 1 + Ksv[Q]) using emission spectraldata is given in Ksv value suggests a moderateaffinity of the compound to EB-bound CT-DNA and that it can competitivelydisplace EB from DNA via an intercalative mode of binding /I = log Kbin + n log[Q]. The bimolecular quenching rate constant(Kq) was calculated according to the equation Ksv = Kqτ0 as 1.88 × 1011 M–1·s–1. This value is higher than those of typical dynamicquenchers (∼1010 M–1·s–1).63 According to the result, EB was displaced from CT-DNAstatically instead of dynamically.64A typical linear Scatchard plot for compound The compound’s ability to displace EB and bind with CT-DNAwas agreed with electronic absorption spectral data and indicatedthat the DNA binding mode of the compound was intercalation. Additionally,a molecular docking study was performed to gain insights into themechanism and mode of DNA binding, and the docking results confirmedthe experimental values pertaining to DNA binding studies.2.765 Fluorescencequenching and UV–vis absorption studies have been performedin order to understand the interaction mechanism between compound 3 and BSA.Because of thestructural similarity between bovine serum albumin and human serumalbumin, drug interactions involving BSA are currently the subjectof pharmaceutical studies.66 After additionof compound 3, BSA absorption decreases with a slightblue shift (Δλ = ∼1 nm), indicating a possiblestatic interaction between compound 3 and BSA . The emission spectra were monitored in the300–450 nm range after excitation at 280 nm. 3 was gradually added, indicating that compound 3 quenchedthe fluorescence of BSA as a result of interactions.Fluorescence spectroscopy experiments were conducted with5 μMBSA and varying concentrations of compound Ksv, has beendetermined using the Stern–Volmer equation and the I0/I versus [Q] plot /I] = log Kbin + n log [Q], was used to calculate the binding constant, where Kbin is the binding constant of 3 with BSAand n is the number of binding sites /I versus log [Q] plot, which is given in The quenching constant, [Q] plot 14b in orng sites 5. The van supports the existence of a single binding site in BSAfor compound 3.68 The valuesof Ksv and Kbin for compound 3 further suggested that the compoundinteracts moderatelywith BSA. The bimolecular quenching constant, kq, was also computed using the equation Ksv = kqτ0, andthe values of kq and n are shown in kq (∼1013 M–1·s–1) of compound 3 is higher than the maximum scatter collision quenching constantof diverse kinds of quenchers for biopolymer fluorescence (2 ×1010 M–1·s–1),indicating the existence of the static quenching mechanism.69 Furthermore, ΔG was calculatedas −24.75 kJ·mol–1 and showed spontaneityof compound 3 binding with BSA.According to the Scatchard equation, the value of 2.8Table S1), so we performed the remaining docking studies using thatmethod in this study. We used two differently shaped DNA molecules.In the first one, the intercalator distorted the DNA  in one of the chains, suggesting that compound 3 can bind and be carried by BSA. On the other hand, the bindingenergy of compound 3 was comparable to that of naproxen in the other chain of BSA. The interactions formed between naproxenand BSA in the crystal structure and docked models are shown in Figure S7.We also performed docking studies on compound 2.92.9.13 can inhibit cell proliferationin three cancer cell lines  and onenormal cell line (HEK-293). Under identical conditions, the conventionalanticancer drug etoposide was used as a positive control, and cisplatinwas selected as parallel control.The MTT assay was usedto determine whether compound 3 wasfound to have higher cytotoxic activity than etoposide for all celllines in a dose-dependent manner after 24, 48, and 72 h. Compound 3 was found to be more cytotoxic than cisplatin in the Caco-2and LNCaP (for 48 and 72 h) cell lines  against Caco-2 cells for 24, 48, and 72 h were lower thanthose of cisplatin  and etoposide , respectively. For LNCaP cell lines, compound 3 showed better or equivalent inhibitory effects [IC50 values are 17.08 ± 0.26 (48 h) and 10.05 ± 0.03 (72 h)]than the cisplatin effects [IC50 values are 25.95 ±0.48 (48 h) and 10.33 ± 0.28 (72 h)]. This suggests that compound 3 could inhibit cell growth in a dose- and time-dependentmanner (75 cytotoxicity of compound 3 was effective as a potentcytotoxic agent.Compound ll lines 17. The Itmanner 7. Compar3 and controldrugs (cisplatin and etoposide) for HEK-293 suggests that compound 3, cisplatin, and etoposide are all cytotoxic against HEK-293cells, and compound 3 is less cytotoxic than the controlcisplatin but more cytotoxic than the control etoposide. These resultsare noteworthy for calculating the selectivity index (SI) of the compoundsstudied against normal cells and cancer cells and predicting theirtherapeutic potential. The term selectivity index refers to how selectivelythe compounds can destroy cancer cells compared to normal cells. HighSI values mean that cancer cells will be killed more quickly thannormal ones.Furthermore, the cytotoxicity of compound 3, etoposide,and cisplatinwere calculated as SI(HEK/Caco-2) = 1.46, 1.10, 0.14, SI(HEK/MDA-MB-231)= 2.30, 0.94, 2.15, and SI(HEK/LNCap) = 1.43, 0.95, 0.18, respectively,showing that the cytotoxic selectivity of compound 3 washigher than those for both etoposide and cisplatin. Compared withthe positive control of cisplatin, the SI of compound 3 was 10 times higher than that for Caco-2 cells and 8 times higherthan that for LNCap cells  stainingin cell culture media by flow cytometry to investigate whether apoptosisaccounted for the cytotoxicity of compound 3 on Caco-2cells. The cells exposed to compound 3  showed high levels of Annexin V and PI positive stainingcompared to the control (without compound 3), suggestingthe induction of cell death by apoptosis  for 48 h, the percentages of apoptosison Caco-2 cells were 2.8, 46.09, 57.77, and 70.25%, respectively.The Annexin V/PI result indicated that compound 3 couldinduce apoptosis of cells with concentration dependence.As shown in 2.9.33, such as MMP and ROS. In many cells, morphologicaland molecular changes in mitochondria are crucial stages in apoptosis.By staining Caco-2 cells with the intrinsically fluorescent dye TMRE,we were able to detect a loss of mitochondrial membrane potentialafter 48 h of treatment with compound 3. According tothe results, compound 3 caused a significant increasein MMP loss in Caco-2 cells in a dose-dependent manner  was synthesized using an azide and alkyne compoundvia click chemistry. In addition to various spectroscopic methods,structural confirmation of the compound was carried out by SCXRD,which revealed a monoclinic system with the P21/c space group. HS analysis was also applied,and van der Waals interactions and hydrogen bonding were found tobe the dominant types of interactions. The volume of crystal voidsshowed that there was no large cavity in the crystal packing. Theinvestigation of the electrostatic, dispersion, and total energy frameworksindicates that stabilization is dominated by the dispersion energycontribution.In the current study, anew 1,4-disubstituted 1,2,3-triazole derivative and both polar and hydrophobic interactions viaBSA and both bindings were spontaneous as we obtained negative ΔG values. In vitro MTT cytotoxic activities of the compoundgave two times better cytotoxicity activity after 48 h in human colorectaladenocarcinoma cell line Caco-2 (16.63 ± 0.27 μM) thancisplatin (32.69 ± 0.87 μM) and nearly three times betterthan etoposide (53.81 ± 0.17 μM). Also, it had the leasteffect on human normal cell line HEK-293 compared to the control drugcisplatin. These findings reflect that the SI values of compound 3 were higher than those for both etoposide and cisplatinon all cancer cells. In addition, compound 3 inducedapoptosis in the Caco-2 cancer cell line. The wet lab and computationalinvestigations suggest that the 1,2,3-triazole derivative (3) may be utilized as an alternative cytotoxic agent for cancer therapywith no adverse effects on healthy cells. However, these results mustbe validated in vivo, and further experiments are needed.In vitro and in silico investigations on DNA/BSAbinding activityof compound 44.11) and 4-azido-1,2-dichlorobenzene (2)were synthesized according to previously reported methods,77 and their detailed syntheses and characterization data are givenin the Supporting Information (SI).All reagentswere purchased from Merck/Sigma. Working solutions were prepared usingdoubly distilled water. A LECO 932 CHNS analyzer was used for microanalysis, and a Bruker 400 MHz spectrometer was used to perform NMRanalysis. IR spectra were recorded with a Nicolet iS10-ATR (Thermo-Scientific).DNA and BSA interaction studies were performed using a T80+ UV–visspectrophotometer (PG) and a SpectraMax i3x Multi-Mode microplatereader (Molecular Devices). X-ray data collections were performedwith a Bruker APEX II QUAZAR three-circle diffractometer using MoKα radiation (λ = 0.71073 Å). 2-Hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde2·H2O  and sodium ascorbate  were addedto a solution of 2-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde  and azide derivative (1.20 mmol) in 10 mL of a 1:1 mixtureof H2O/CH2CI2. After stirring for8 h at RT, the mixture was diluted with 10 mL of H2O/CH2CI2 (1:1), and the organic layer was separated,washed with water (1x), 1 M EDTA (3x), and brine (2x), dried overNa2SO4, and filtered. The solvent evaporatedto give a brownish or yellowish residue. Then, the residue was dissolvedin the minimum amount of CH2CI2, and an off-whiteproduct was precipitated by adding 50 mL of n-hexane.The pure compound was filtered and dried, and single crystals suitablefor X-ray analysis were obtained by the recrystallization of the compoundfrom a methanol/ethyl acetate mixture at room temperature (The compound was prepared according to a previously reported methodby us with minor modifications.perature 1.4.2.1–1): 3155 (C–H)tz, 3074–2837 (Ar–H),1218 (C–O), 1644 (C=O). 1H NMR  δ (ppm): 11.46 , 9.74 , 8.08, 7.91 , 7.62 , 7.47 ,6.64 , 6.56 , 5.33 . 13C APT NMR  δ (ppm): 194.69, 165.15, 164.49, 144.38,135.93, 135.68, 134.27, 133.40, 131.72, 122.50, 121.10, 119.66, 115.82,108.56, 101.92, 62.19. Anal. calcd. for C16H11Cl2N3O3: C, 52.77; H, 3.04; N, 11.54.Found C, 52.65; H, 3.05; N, 11.54.Yield:89%. mp: 144–145 °C. FTIR , 0.97 Å (for CH2), and 0.96 Å (forCH3) and refined using a riding model with the constraintsof Uiso(H) = k X Ueq (C), where k = 1.5 for methyl H atoms and k = 1.2for other H atoms. Crystallographic data for the structure describedin this paper have been deposited with the Cambridge CrystallographicData Centre as Supporting Information,CCDC No. 2240671.The crystallographicdata were processed by SHELX program packages  adduct. The excitation wavelength wasfixed to 510 nm, and the emission spectra were recorded (λem = 500–700 nm). The quenching efficiency ofthe compound was analyzed using the Stern–Volmer and Scatchardequations, and the Stern–Volmer (quenching) constant, Ksv, the bimolecular quenching rate constant, kq, binding constant, Kbin, and the number of binding sites per nucleotide, n, were calculated.84The competitiveemission quenching experiment was carried out usinga well-known intercalating agent and fluorescent probe for DNA, ethidiumbromide (EtBr).SI.Thedetailed experimental procedures including solution preparationsand calculations of the binding data for both absorption studies andfluorescence studies are provided in the 4.53 (10 μM)] at room temperature in 10mM Tris-HCl buffer with a pH of 7.4.85The BSA–compoundinteractions are studied by comparing the UV–vis spectra (200–400nm) of BSA (10 μM) alone and a mixture of [BSA (10 μM)+ compound SI.BSA fluorescence spectral studies of the compound and related calculationswere the same as those performed for the [CT-DNA + EB] quenching methoddescribed in 4.63 , we first investigatedthe performance of different docking schemes to identify the bestone in terms of docking performance, which was described by its capabilityin terms of placing the ligand into its position in the crystal. Towardthis end, we used standard precision and extra precision docking schemes,both of which are implemented in Schrodinger software.86 The schemes with enhanced sampling are namedSP2 and XP2. On the other hand, when we used a scheme that containedboth enhanced sampling and enhanced planarity, we named the methodsas SP3 and XP3. We achieved more accurate results with the XP3 method,wherein the enhanced planarity of conjugated pi groups was implemented.In docking, we used crystal structures with PDB IDs: 1Z3F,873FT6,881D30,89 and 8EC1.90 The first two structures can be given as examples of intercalation,whereas the last two represented the groove binding. We performeddocking using the OPLS3e force field91 anddescribed the box dimensions to include the whole DNA present in thecrystal structures studied. The molecules other than the ligand andDNA, which were present in the crystal, were removed before performingdocking studies. The ionization state of the ligands and DNA was assignedat pH 7.4.As we opt to examine the binding mode of compound 3 using the same docking scheme, namely, XP3, that we usedin DNA docking. There is a couple of crystal structures pertainingto the complex of BSA with different ligands in the PDB. Among them,we used the crystal structure of naproxen-bound BSA (PDB ID: 4OR0)92 and took naproxen from that structure, while the structureof BSA was obtained from the crystal structure pertaining to the apoform of the protein (PDB ID: 3V03).93 The ionization stateof the ligand and protein was assigned at pH 7.4, and the OPLS3e forcefield91 was used. Disulfide bonds wereadded to the protein except for Cys34 in correspondence with its physiologicalstate.93 The calcium ions and water moleculesthat were present in the crystal structure of BSA were kept duringdocking calculations.We also performed docking studies on BSA and compound 4.72) and cultured under standard conditions in Dulbecco’s modifiedEagle medium (DMEM), 10% heat-inactivated fetal bovine serum, 100U mL–1 penicillin, 100 U mL–1 streptomycin,and 4 mM l-glutamine. Compound 3 (10 mM in DMF),clinically used cisplatin , and etoposide(10 mM in DMF) formulation was used as stock solution in experiments.A cell culture medium was used to make further dilutions, while keepingthe equivalent of DMF below 0.5% of the total volume.94Human cancer cell lines and a human healthy cell line (HEK-293)were obtained from the European Collection of Cell Cultures. The cellswere incubated in a humidified incubator (37 °C, 5% COSI.The detailed procedures of the cell viability inhibitionassay, Annexin V/PI double-staining assay, MMP, and ROS determinationstudies are provided in the


The higher efficiencies of the active sites of S.C to adsorb more CFX molecules (n = 2.08–2.31) than N.C (n = 1.41–2.16) illustrate its higher adsorption capacity. The energetic investigation [adsorption (˂40 kJ/mol) and Gaussian (˂8 kJ/mol) energies] suggested adsorption of CFX by N.C and S.C mainly by physical processes such as van der Waals forces, hydrogen bonding, dipole bonding, and π–π interactions. Moreover, the determined thermodynamic functions including entropy, internal energy, and free enthalpy reflect the spontaneous and endothermic uptake of CFX on the surfaces of N.C and S.C.A sub-bituminous natural coal sample (R.C) was treated with sulfuric acid (S.C) and nitric acid (N.C) as modified products and enhanced adsorbents for obtaining ciprofloxacin (CFX) antibiotic residuals from water. The characterization studied demonstrates enhancement in the surface area and the incorporation of new active oxygenated, sulfur-bearing, and nitrogen-bearing chemical groups into the structure of coal samples. This was reflected in the adsorption capacities that were enhanced from 164.08 mg/g (R.C) to 489.2 mg/g and 518.5 mg/g for N.C and S.C, respectively. The impact of the acid modification processes was evaluated based on the energetic and steric properties of their adsorption systems considering the parameters of the advanced monolayer equilibrium model with one energy site. The determined occupied active sites’ density of R.C (46.32–61.44 mg/g), N.C (168.7–364.9 mg/g), and S.C (159.2–249.9 mg/g) reflects an increase in the quantities of active centers after the acid treatment processes, especially with HNO In the later decades, pollution of water sources with different species of organic pollutants was reported extensively as an associated result of the increase in chemical and pharmaceutical industries . Pharmac17H18FN3O3) (CFX) is one of the fluoroquinolone analog antibiotics applied widely during the treatment of infectious diseases  and as an antibacterial agent against Gram-negative and Gram-positive bacilli , and enhancement in the surface area . OxidatiHowever, the efficiency of acid oxidation or modification of coal is controlled by the oxidation conditions, chemical activators used, and the concentrations of the used acid . Unfortu2SO4)  and nitric acid (HNO3)  were applied during the oxidation and modification of the coal sample, respectively. CFX (C17H18FN3O3) fluoroquinolone antibiotic (≥98% (HPLC); Sigma-Aldrich; Egypt) was used as a source of antibiotic residuals during the adsorption experiments.The coal used during the acid oxidation processes is sub-bituminous coal and was obtained from El-Maghara coal mine in Sinai, Egypt. The chemical composition of the used coal was detected based on the ultimate-proximate analyses and presented in 2SO4  and HNO3  at a certain temperature (150°C) for 60 min. Then, samples modified by the two acids were washed extensively with distilled water to neutralize the surfaces of the oxidized products and remove the excess in the applied acids during the treatment processes. Finally, the products were dried for 10 h at 65°C using a digital dryer, kept in containers, and labeled as S.C  and N.C .First, the collected raw coal particles were ground by using a normal home blender to reduce the size of the coal fractions to determined range from 20 μm to 70 µm. After that, the obtained ground coal (10 g) was homogenized within two separate beakers of Ho to 80o. During the analysis, the diffraction data were recognized after exposing the samples as a powder to the X-ray radiation source at 1.5418 Å as the measuring wavelength (λ). The used X-ray is Cu–Kα–X-ray and the producing source is Cu anode, which is provided with 40 mA as the operation current and 40 kV as the determination voltage. The oxidation effects of the acids and the incorporated new chemical groups were identified based on the resulting Fourier Transform Infrared (FT-IR) spectra of the treated structures using an FT-IR spectrometer (FTIR−8400S) in the transmission mode within an estimation range from 4,000 to 400 cm−1. During the FT-IR investigation, the modified samples were milled with KBr powder at an adjusted ratio of 1:100, and then the mixtures were compressed by using a hydraulic press into pellets which were fixed in the sample holder of the FT-IR spectrometer. The morphologies of the acid-treated coal products in comparison with those of the raw sample were described according to their SEM images using a scanning electron microscope . Also, the impact of the treatment reactions on the surface area of the oxidized coal products was followed based on the recognized N2 nitrogen adsorption/desorption isotherm curves by using the Beckman Coulter surface area analyzer (SA3100 type) after degassing for 15 h at 105°C and the measuring temperature was adjusted at 77 K. The values of the specific surface area were calculated according to the Brunauer–Emmett–Teller (BET) equation, while the porous properties were calculated according to the Barrett–Joyner–Halenda (BJH) method.The impact of acid modification on the physicochemical properties of coal was assessed in terms of the changes in the structural, morphological, chemical, and textural properties. The structural properties of coal, S.C, and N.C were evaluated according to their XRD patterns using an X-ray diffractometer ) within the estimation range for the 2 Theta angle from 5The adsorption of CFX by S.C and N.S in comparison with raw coal was accomplished in the batch form considering the experimental effects of the essential parameters such as the pH (pH 2 to pH 9), adsorption duration , and the CFX starting concentrations (100 mg/L to 800 mg/L) considering the change in the adsorption temperature from 298 K to 318 K. Moreover, the coal dosages and the tested CFX volumes were assessed at fixed values of 0.2 g/L and 250 mL, respectively. All these adsorption experiments were repeated for three runs, and the obtained average results were used for plotting of the results. The remaining CFX concentrations after conducting the desorption experiments were measured by the HPLC system (Merck/Hitachi) after the removal of coal particles. The applied HPLC system during the determination processes consists of a Luna column (150 mm × 4.6 mm), a pump (L-7100), and a detector (L-7400) in addition to the injection valve (Rheodyne 7725i), which is attached to the sampling loop (20 mL). The column  of the used HPLC system consisted a stationary phase composed of PFP with 5 mm thickness. During CFX determination, a prepared mixture of distilled water and analytical grade methanol (60/40 volume ratio) was used as the mobile phase and introduced to the systems at an operative flow rate of 1 mL/min and 10 µL as an injection volume.e) according to Eq. o, Ce, V, and m are the starting CFX concentration (mg/L), remaining CFX concentration (mg/L), tested volume (mL), and the dosage of coal dosage (mg), respectively.The measured remaining CFX concentrations were applied during the calculation of the actual or the experimental CFX adsorption capacities (QR2) (Eq. 2) (Eq. R2) and the recognized root mean square error (RMSE)  Eq. . Regardi. 2) Eq. , non-linMSE) Eq. . The m′,o–30o (002) and 40o–50o (101), respectively (2SO4 (S.C), the resulting pattern reflected deviation in the first peak (10o–32o) and significant reduction in the second peak ; aliphatic CH, CH2, and CH3 ; C-H in the methyl group ; C-H in the methylene group ; C-O group ; C=C ; -C=O (1716 cm-1); and hydroxyl groups  (−1) and reorganization of the new band related to the carboxylic groups (C=O) (1712 cm−1) , symmetric O=S=O , asymmetric O=S=O , and C-S (578 cm−1) (−1). Additionally, there is a significant reorganization for the new band of the C=O stretching  and the incorporated nitrogen-bearing groups    (Shaban 00 cm−1) . Regardi12 cm−1)  are obse12 cm−1) . Moreove78 cm−1)  (Fonseca78 cm−1) . The obt.9 cm−1)  (Alvarez.9 cm−1) . This al2SO4 than that of HNO3 , 2–86 nm (N.C), and 2–80 nm (S.C)) and the estimated average pore diameters , 34.8 nm (N.C), and 22.4 nm (S.C)). The increase in the pore volume is related to the significant increment in the micropores as a result of the dissolving and leaching of entrapped metal and carbonate impurities. Additionally, the changes in the topography of the coal particles strongly affected the determined values of pore diameters and pore volumes. Such enhancement in the pore volumes and formation of micropores results in a significant enhancement in the surface area of coal (5.4 m2/g), which increased to 26.4 m2/g and 18.3 m2/g after its structural modification with H2SO4 and HNO3, respectively, which can strongly affect the adsorption properties  values of R.C (6.6), N.C (6.2), and S.C (5.4), which demonstrate the high saturation of their surfaces with negative charges beyond these values.The solution pH strongly affected the adsorption affinities of the studied adsorbents toward the dissolved ions either by controlling the ionization behaviors of the dissolved ions or by controlling the surficial charges of the used solid adsorbents. The changes in the CFX adsorption properties of raw coal (R.C), S.C, and N.C with the adjusted pH of the solutions were addressed from pH 2 to pH 8. The essential experimental factors were considered at fixed values , 240 min (adsorption duration), 200 mg/L (CFX concentration), 250 mL (volume), and 298 K (adsorption temperature)). The observed CFX uptake results validate the enhancement with adjustable increments in the pH of the solutions until pH 8 (66.4 mg/g (R.C), 82.3 mg/g (N.C), and 90.6 mg/g (S.C)) . At pH 9The changes in the CFX adsorption properties of R.C, N.C, and S.C by increasing the uptake duration were followed from 30 min to 180 min. The essential experimental factors were considered at fixed values , pH 8 (adsorption pH), 200 mg/L (CFX concentration), 250 mL (volume), and 298 K (adsorption temperature)). There are significant variations in the actual CFX adsorption rates by R.C, N.C, and S.C, with increasing the adsorption duration as observed evidently in the segmental forms of the presented curves . First, The intra-particle diffusion properties of R.C, N.C, and S.C as the used adsorbents of CFX reflected segmental behaviors . This isR2) and Chi-squared (χ2) were presented as the controlling factors of the fitting degrees of the non-linear fitting processes with the two assessed kinetic models , 102.5 mg/g (N.C), and 127.8 mg/g (S.C)) and theoretically obtained values as a parameter of the PFO model (92.25 mg/g (R.C), 106.7 mg/g (N.C), and 129.3 mg/g (S.C)) ) . Howeverg (S.C)) . Both phg (S.C)) .max values of them.The experimental effect of the CFX starting concentration on the determined uptake capacities was assessed from 100 mg/L to 800 mg/L to denote the actual maximum capacities of R.C, N.C, and S.C, as well as the equilibrium properties of their adsorption systems. The essential experimental factors were considered at fixed values , pH 8 (adsorption pH), 1,080 min (adsorption duration), and 250 mL (volume)), while the adsorption temperature studied was within the range 298 K–318 K. The determined results experimentally validate the enhancement in the CFX uptake properties of R.C, N.C, and S.C by testing the adsorption of the antibiotic molecules at high starting concentrations . This enmax values of CFX using R.C are 132 mg/g (298 K), 146.7 mg/g (308 K), and 161.8 mg/g (318 K) (The obtained Q (318 K) . These v (318 K) , while t (318 K) . These dR2 (determination coefficient) in addition to χ2 (Chi-squared)   . The adssquared) . Accordisquared) . Moreove (318 K) .The evaluation of the D–R equilibrium model strongly signifies the heterogeneity of the apparent energy during the adsorption reactions, either by the homogenous or heterogeneous surfaces of the used adsorbents . Moreovem), number of adsorbed ions per site (n), and saturation adsorption capacity (Qsat)) and energetic parameters , adsorption energy (ΔE), free enthalpy (G), and entropy (Sa))  fit strongly with the monolayer model with one energy site ) . The fitrgy site ; Table 4CFX), which were adsorbed per each free and active uptake site of R.C, N.C, and S.C, significantly validate the orientation of the adsorbed CFX molecules in addition to the affected mechanism. The obtained values of nCFX using R.C (n = 2.647–2.90), N.C (n = 1.41–2.16), and SS.C (n = 2.08–2.31) are higher than 1 . This behavior validates a considerable decrease in the CFX aggregation properties during the uptake reactions using R.C, N.C, and S.C, as well as the high CFX adsorption rates before the aggregation of the molecules ) on the surfaces of R.C, N.C, and S.C significantly reflects the quantities of the effective active sites and validates the impact of the performed acid treatment processes either by HNO3 or by H2SO4 ((CFX) values of R.C are 46.32 mg/g (298 K), 56.33 mg/g (308 K), and 61.44 mg/g (318 K). These values enhanced significantly after the modification of coal with HNO3 (168.7 mg/g (298 K), 224.88 mg/g (308 K), and 364.9 mg/g (318 K)) and H2SO4 (159.2 mg/g (298 K), 188.55 mg/g (308 K), and 249.9 mg/g (318 K)) (3 and H2SO4) as chemical activators on the surface of coal in addition to the significant incorporation of new forms of oxygenated, nitrogen-bearing, and sulfur-bearing chemical groups on its structure. The significant increase in the Nm (CFX) values of R.C, N.C, and S.C in terms of the evaluated temperature from 298 K to 318 K was attributed to the observed decrease in the achieved nCFX values. The increase in nCFX values or the aggregation properties of CFX molecules resulted in a detectable decrease in the predicted numbers of the effective sites which were occupied during the captured antibiotic molecules ) ; Table 4olecules . Moreoveolecules . This misat) validates the most appropriate theoretical values of the maximum capacities of the studied products as adsorbents in comparison with the detected values based on the evaluated classic isotherm models. The Qsat values of CFX estimated by R.C are 134.33 mg/g (298 K), 152.09 mg/g (308 K), and 164.04 mg/g (318 K). The estimated Qsat values increased at a remarkable rate after the modification of coal with HNO3 (364.5 mg/g (298 K), 398.03 mg/g (308 K), and 489.24 mg/g (318 K)) and H2SO4 (367.9 mg/g (298 K), 401.6 mg/g (308 K), and 518.5 mg/g (318 K)). Considering the high dependence of the Qsat values on the estimated nCFX and Nm(CFX) values, the remarkable enhancement in the estimated Qsat of CFX by N.C and S.C can be attributed to the previously reported increase in the quantities of the effective sites by incorporating additional chemical groups as well as the reported enhancement in the surface area. However, the samples treated by HNO3 (N.C) exhibit higher Nm(CFX) values than the samples treated by H2SO4 (S.C), which shows lower Qsat values. This might be due to the higher surface area of S.C than of N.C in addition to the efficiency of the S.C adsorption sites to adsorb high numbers of CFX molecules than N.C.The adsorption capacity of CFX at the saturation states of R.C, N.C, and S.C  can interpret the adsorption mechanism  that affects the adsorption of CFX by R.C, N.C, and S.C. For chemical adsorption, (ΔE > 80 kJ/mol), while for physical adsorption, (ΔE  kJ/mol) . The adsforces 4–0 kJ/mol, kJ/mol) .∆E=RT lThe ΔE values of CFX calculated by R.C are 3.49 kJ/mol (298 K), 3.49 kJ/mol (308 K), and 3.37 kJ/mol (318 K). The estimated values of N.C are 4.82 kJ/mol (298 K), 4.32 kJ/mol (308 K), and 4 kJ/mol (318 K), while the obtained values during the adsorption of CFX by S.C are 4.51 kJ/mol (298 K), 3.81 kJ/mol (308 K), and 3.89 kJ/mol (318 K) . These v(CFX), and C1/2  (The entropy (Sa) properties of CFX adsorption systems by R.C, N.C, and S.C display the disorder and order properties of the surfaces of coal-based adsorbents at different concentrations of the adsorbates and different adsorption temperatures. The values of Sa can be calculated according to Eq. uration) .SaKB=NmThe calculated values of Sa of the CFX adsorption reactions by R.C, N.C, and S.C decrease at considerable rates due to their high concentrations in the solutions . Therefoint) and free enthalpy (G) properties of CFX adsorption processes by R.C, N.C, and S.C were evaluated based on the calculated values from Eqs.(CFX), and C1/2  in addition to the value of translation partition (Zv)  .EintKBTint values of CFX adsorption reactions by R.C, N.C, and S.C exhibit negative signs and increase regularly with the temperature ranging from 298 K to 318 K. Based on these results and behaviors, the R.C, N.C, and S.C adsorption systems of CFX show spontaneous and endothermic behaviors . The reusability tests validate significant stabilities of R.C, N.C, and S.C as adsorbents of CFX residuals from water. The determined CX uptake capacities during the recyclability of R.C are 161.8, 156.3, 148.2, 135.3, and 122.8 mg/g for runs 1–5, respectively . For N.C−, and–SO3H in addition to other structural chemical groups and the incorporated nitrogen and sulfur chemical groups  or sulfuric acid (S.C) contain several species of active oxygenated chemical groups such as–COOH, –OHl groups . Considel groups . The firl groups .2SO4- and HNO3-modified coal products were prepared as enhanced and low-cost adsorbents of ciprofloxacin residuals. The modified coal samples exhibit enhanced CFX adsorption capacities (N.C (489.2 mg/g) and S.C (518.5 mg/g)) as compared to raw coal (164.08 mg/g). The adsorption systems, in terms of adsorbate/adsorbent interactions, were illustrated based on the steric and energetic properties. Based on the CFX-occupied active site’s density (Nm as compared to R.C (61.44 mg/g). The observable higher capacities of the actives sites on the surface of S.C to adsorb more CFX molecules (n = 2.08–2.31) than N.C (n = 1.41–2.16) illustrate the determined higher adsorption capacity. The values of the adsorption (˂40 kJ/mol) and Gaussian (˂8 kJ/mol) energies demonstrate the dominant effect of the physisorption mechanism, and the thermodynamic function validates the spontaneous and endothermic properties of these processes.H


L-theanine (LT), which is a major amino acid found in green tea, was shown to alleviate Vincristine (VCR)-induced peripheral neuropathy and associated neuronal functional changes in rats. To induce peripheral neuropathy, rats were administered VCR at a dose of 100 mg/kg/day intraperitoneally on days 1–5 and 8–12, while control rats received LT at doses of 30, 100, and 300 mg/kg/day intraperitoneally for 21 days or saline solution. Electrophysiological measurements were taken to evaluate the nerve functional loss and recovery through motor and sensory nerve conduction velocities. The sciatic nerve was examined for several biomarkers, including nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), total calcium, IL-6, IL-10, MPO, and caspase-3. The results showed that VCR caused significant hyperalgesia and allodynia in rats; decreased nerve conduction velocity; increased NO and MDA levels; and decreased GSH, SOD, CAT, and IL-10 levels. LT was found to significantly reduce VCR-induced nociceptive pain thresholds, decrease oxidative stress levels , increase antioxidative strength , and reduce neuroinflammatory activity and apoptosis markers (caspase-3). LT’s antioxidant, calcium homeostasis, anti-inflammatory, anti-apoptotic, and neuroprotective properties make it a potential adjuvant to conventional treatment in VCR-induced neuropathy in rats. Chemotherapy-induced neuropathic pain is a prevalent adverse effect of cancer treatment caused by various chemotherapeutic medications, such as vinca alkaloids, platinum medicines, and taxanes . This coConventional analgesics and antineuralgic drugs provide limited relief and are restricted due to side effects ,3,4. The+ [L-theanine (LT) is a molecule with a similar structure to glutamate and glutamine, accounting for 0.5–2% of the dry weight of tea leaves. LT consists of ethylamine and glutamate and is rapidly absorbed into the circulation of the gastrointestinal system and subsequently distributed to various organs, including the brain, via cotransport with Na+ . LT was + ,10. Addi+ ,10,11, a+ ,13,14,15In this study, we aimed to investigate the potential of LT in alleviating VCR-induced neuropathy in rats using a well-established model. Various assessments were carried out, including the Von Frey hair test, acetone drop test, and hot plate test to evaluate the degree of neuropathy. Additionally, electrophysiological investigations were conducted to examine sensory and motor nerve conduction velocities (SNCV and MNCV). The levels of nitrite (NO) and malondialdehyde (MDA) were measured to determine the nitrosative and oxidative statuses, while glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) enzyme activities were analyzed to evaluate the antioxidant power. Total calcium, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), myeloperoxidase, and severe neurodegeneration were also examined, as they were strongly associated with VCR-induced neuropathy in animal models ,5,6,7,8.2 overdose at the end of the study.Male Wistar rats weighing between 220 and 250 g  were utilized in this study. The animals were housed in Plexiglas cages in groups of three with ad libitum access to food and water under controlled temperature conditions (22 ± 3 °C) and a 12 h light/dark cycle with the lights turned on at 7:00 a.m. All study procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University College of Medicine and College of Public Health, following the guidelines published by the National Institutes of Health of the United States . The study tests were conducted during the light phase of the cycle, and the animals were euthanized using a COPhysiological saline and distilled water were used to prepare Vincristine sulfate salt  and N-ethyl-L-glutamine  for the study. The doses used in the study were based on previous research . LT was For this study, male Wistar rats were randomly divided into eight groups with eight rats in each group: control (C), LT 30 mg/kg treatment (T30), LT 100 mg/kg treatment (T100), LT 300 mg/kg treatment (T300), VCR treatment (V), VCR + LT 30 mg/kg treatment (V + T30), VCR + LT 100 mg/kg treatment (V + T100), and VCR + LT 300 mg/kg treatment (V + T300). The control group received an intraperitoneal (i.p.) injection of normal saline solution. The T30, T100, and T300 groups received i.p. injections of LT at doses of 30, 100, or 300 mg/kg, respectively, for 21 days. The V group received i.p. injections of 100 μg/kg VCR for two 5-day cycles with a 2-day pause (days 1–5 and 8–12) and normal saline i.p. on days 1–21. The V + T30, V + T100, and V + T300 groups received i.p. injections of 100 μg/kg VCR for two 5-day cycles with a 2-day pause (days 1–5 and 8–12) and LT i.p. at doses of 30, 100, or 300 mg/kg, respectively, on days 1–21. VCR was administered 60 min before the LT or normal saline injection on days 1–5 and 8–12. Behavioral parameters were measured before the VCR administration on day 1 and days 7, 14, and 21 after each LT or normal saline injection. Nerve conduction velocities were measured after the behavioral examinations on day 21 for electrophysiological studies. All biochemical studies were conducted after the electrophysiological experiments were completed see . During We evaluated the pain sensitivity of the hind paws of rats for thermal hyperalgesia using Eddy’s hot plate , as per the method described by Eddy et al. , with ceTo evaluate the sensitivity of rat hind paws to chemical allodynic pain, we used an acetone drop application following the protocol of Yoon et al. , with a By increasing the pressure on the left hind paw, we measured the mechanical nociceptive threshold in grams . The mechanical nociceptive threshold was determined by withdrawing the left hind paw. The cutoff pressure was kept at 450 g .The mid-plantar surface of the left hind paw was tested for mechanical allodynia using calibrated nylon filaments  of varying stiffnesses. Each filament was applied to the paw 10 times, beginning with the softest and increasing in stiffness. A positive response was recorded if the left hind limb was quickly withdrawn. To determine the threshold value in grams, the filament was applied five times out of ten trials (50% response). A cut-off pressure of 30 g was used .After assessing various behavioral parameters on day 21, the animals were anesthetized for electrophysiological studies of motor and sensory nerve conduction velocities.The student physiograph  and biopotential coupler and stimulator device were used to evaluate the motor nerve conduction velocity (MNCV) as per Thomsen et al.  and SainZ, and 10 pulses per train of intensity current were applied to the middle (3rd) digital nerve of the hind leg, resulting in a maximum amplitude response. The peak-to-peak amplitude was subsequently measured. The recording needles were maintained at a distance of 10 mm apart, and there was an approximate 25 mm separation between the recording and stimulation electrodes. The body temperature of the rats remained at a constant 37 °C throughout the study. Latencies were measured during the stimulus–response interval, and the maximal sensory nerve action potential was calculated using the following formula: the sensory nerve conduction velocity, also known as the H-reflex, was determined by dividing the onset of latency by the peak of the initial negative deflection (distance between stimulating and recording electrodes).A modified technique, as described by Kurokawa et al. , was uti2 chamber was utilized to minimize stress. Both legs’ sciatic nerves were immediately isolated, and the nerve was excised distal and proximal to the point of transection. Exactly beneath the point of transection of the sciatic nerve, a 1 cm diameter piece of tissue was removed. The samples were frozen and subsequently analyzed together after freezing. A sciatic nerve homogenate  was prepared using 0.1 M tris HCl buffers (pH 7.4). The homogenates were kept in ice water for 30 min, followed by centrifugation at 2000× g. Proteins, nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) were measured from the clear supernatant. The tissue beneath the sciatic nerve was homogenized and centrifuged at 5000× g for 10 min. The myeloperoxidase (MPO) activity was subsequently measured in pellets following centrifugation.On day 21, following the completion of electrophysiological studies, a COThe protein concentration was estimated using the method developed by Lowry et al. , with bog for 20 min at 4 °C to separate the supernatant. Next, 300 μL of vanadium (III) chloride  was added to 200 μL of the supernatant. The mixture was incubated at 37 °C for 45 min. The absorbance was measured at a wavelength of 540 nm. A standard curve was generated using sodium nitrite [To determine the oxidative status of nitrites in the sciatic nerve samples, 150 μL of the supernatant was dissolved in 0.3 M sodium hydroxide. After 5 min, the precipitated protein was removed by adding 75 μL of zinc sulfate (5%). The mixture was subsequently centrifuged at 15,000×  nitrite . The resg for 10 min. The resulting supernatant was then measured for its absorbance at 532 nm, and standard curves were created using Tetramethoxypropane . The concentration of MDA was expressed as nmol/mg protein.To assess the tissue lipid peroxidation levels, the concentration of MDA was determined by reacting it with TBA, following the procedure outlined in . In shorTo measure the reduced glutathione concentration, equal amounts of sciatic nerve homogenate were mixed with 10% trichloroacetic acid  and centrifuged to separate the proteins. Then, 2 milliliters of phosphate buffer (pH 8.4), 500 milliliters of 2,5-dinitrobenzoic acid, and 400 milliliters of double-distilled water were added to 10 microliters of the resulting supernatant. The mixture was vortexed and the absorbance was measured at 412 nm within 15 min. The concentration of reduced glutathione was reported as μg/mg protein, according to the method described in .v/v mixture of ethanol and chloroform was added to the same volume of sample and centrifuged, and the activity was measured in the ethanol phase of the supernatant. One unit of SOD was defined as the amount required to inhibit 50% of the reduction of nitro blue tetrazolium. The SOD activity was expressed as U/mg protein, according to the method described in [To measure the superoxide dismutase (SOD) activity, xanthine-xanthine oxidase  was used as a superoxide generator to inhibit the reduction of nitro blue tetrazolium. A 5/3 ribed in .2O2; Sigma, USA) at 240 nm, according to the method of Aebi [2O2)/min) at 25 °C, which produced lysate by catalysis. Bovine serum albumin was used as the standard to determine the total protein concentration in the cell lysates [The catalase activity was determined by measuring the absorbance of hydrogen peroxide  in ice-cold conditions, followed by centrifugation at 2000× ribed in .g for 15 min, following the procedure outlined in [To estimate the levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in the nerve samples, the samples were first homogenized in 0.40 mL of ice-cold phosphate buffer saline and centrifuged at 10,000× lined in . The resThe activity of myeloperoxidase (MPO) was determined by reading the standards at 460 nm on a spectrophotometer. One unit of MPO activity was defined as the change in absorbance of 1.0 unit/min at pH 7.0 and a temperature of 25 °C and was calculated from the initial reaction rate with peroxide (1 μM) as the substrate. The results were expressed as U/mg of protein .The levels of caspase-3, which is an executioner molecule in the apoptotic cascade, were determined by cleaving the chromogenic caspase substrate Ac-DEVD-pNA, which is a protease that is rapidly activated when cells are exposed to apoptotic conditions and cleaves poly(ADP-ribose) polymerase. The amount of caspase-3 was measured at 405 nm using a spectrophotometer following the manufacturer’s instructions . The results were expressed as ng/mg protein.p < 0.05.The mean ± standard error of the mean was used to represent all the results. A repeated-measure three-way analysis of variance (ANOVA) was used to analyze the behavioral assessment data. A two-way ANOVA was used to analyze the electrophysiological data and biochemical estimations separately. Tukey’s test was used to compare groups post hoc. Statistical significance was set at p > 0.05; T100 vs. C, p > 0.05; T300 vs. C, p > 0.05).The values of the tested parameters were very similar before the VCR administration. The post hoc analysis of the tested parameters showed no significant differences between the LT groups  and the C groups   (p < 0.001) (21 days: −70%: 13.2 ± 1.4–3.9 ± 0.9 s) (p < 0.001) and acetone drop test (p < 0.001)  (p < 0.001) (21 days: −76%: 11 ± 1.4–2.6 ± 1.1 s) (p < 0.001) compared with the C group.VCR-induced neuropathy; caused significant development in thermal/mechanical hyperalgesia and allodynia. To investigate the ameliorative effects of LT on VCR-induced neuropathy in rats, LT was administered. VCR-induced neuropathy led to the production of persistent thermal hyperalgesia and allodynia with a reduction in response latency at approximately day 7, with this effect peaking on day 14 and lasting through to the end of the experiment for the hot plate test a  compared with the V group in the hot plate and acetone drop tests. However, LT (100 mg/kg) significantly increased the response latency, but only on day 21 of treatment compared with the V group in the hot plate (3.9 ± 0.9–6.4 ± 1.3 s) (p < 0.001) and acetone drop tests (2.6 ± 1.1–5.1 ± 1.2 s) (p < 0.001). In addition, LT (300 mg/kg) increased the response latency significantly in rats compared with the V group on day 14 of treatment in the hot plate and acetone drop tests (hot plate test: 3.5 ± 0.7–5.8 ± 0.9 s) (p < 0.001) (acetone drop test: 2.8 ± 1–5.5 ± 1.4 s) (p < 0.001) and on day 21 (hot plate test: 3.9 ± 0.9–7.8 ± 1.1 s) (p < 0.001) (acetone drop test: 2.6 ± 1.1–7.3 ± 1.6 s) (p < 0.001). These results indicate that the LT treatment prevented the VCR-induced development of thermal hyperalgesia and allodynia.No significant changes were found in the response latency in the V + T30 group  and von Frey hair test (p < 0.001) in the V group. The low PWT of the V group was maintained throughout the study in the paw pressure test  (p < 0.001)  (p < 0.001) and von Frey hair test  (p < 0.001)  (p < 0.001).On day 7, the VCR administration also caused significant development of mechanical hyperalgesia and allodynia as shown by a decrease in the hind paw withdrawal threshold (PWT) in the paw pressure test a  between the V + T30 and V groups. However, the administration of LT (100 and 300 mg/kg) attenuated the VCR-induced decrease in the PWT for mechanical hyperalgesia (14 days—LT 300 mg/kg: 203 ± 11–230 ± 10 g)  (21 days—LT 100 mg/kg: 198 ± 13–227 ± 9 g and LT 300 mg/kg: 198 ± 13–243 ± 15 g)  and allodynia (14 days—LT 300 mg/kg: 3.2 ± 1.2–6.8 ± 1.8 g)  (21 days—LT 100 mg/kg: 4.1± 0.9–7.1 ± 1.5 g and LT 300 mg/kg: 4.1 ± 0.9–7.5 ± 1.5 g)  compared with the V group in a dose-dependent manner from day 14 to 21 after the treatment. These results indicate that the LT treatment prevented the VCR-induced development of mechanical hyperalgesia and allodynia. Based on the above results, our observed NP was verified since the LT treatment ameliorated VCR-induced neuropathy.There were no significant differences in the PWT  (p < 0.001)   a and SNC< 0.001) b on day p < 0.001; V vs. V + T300, p < 0.001) and SNCV (LT 100 mg/kg: 11.6 ± 3.6–21.4 ± 3.9 m/s (32%); LT 300 mg/kg: 11.6 ± 3.6–33.1 ± 5.9 m/s (70%))  in a dose-dependent manner. These results indicate that the LT treatment prevented a VCR-induced loss of neuronal electrical function.The LT administration (100 and 300 mg/kg) attenuated the VCR-induced decreases in MNCV (LT 100 mg/kg: 21.9 ± 5.1–32.9 ± 5.4 m/s (31%); LT 300 mg/kg: 21.9 ± 5.1–45.1 ± 4.7 m/s (65%))  (p < 0.001) , malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) enzyme activities in the sciatic nerve. On day 21, compared with the control (C) group, both the nitrite (2.6 ± 1–28.4 ± 3.8 nmol/mg protein (992%)) (< 0.001) a and MDA< 0.001) b levels p < 0.001 and MDA: p < 0.001) (nitrite: 28.4 ± 3.8–14.9 ± 1.7 nmol/mg protein (−52%) and MDA: 4.3 ± 0.3–2.1 ± 0.2 nmol/mg protein (−73%)) . These results suggest that the LT treatment could effectively inhibit VCR-induced increases in sciatic nitric oxide and lipid peroxide production.However, the treatment with LT at doses of 100 mg/kg and 300 mg/kg significantly inhibited the elevated nitrite and MDA levels in the V groups (nitrite: 28.4 ± 3.8–18.4 ± 2.4 nmol/mg protein (−39%) and MDA: 4.3 ± 0.3–2.9 ± 0.3 nmol/mg protein (−47%))  (p < 0.001) (p < 0.001) ) (< 0.001) a, SOD (1< 0.001) b, and CA< 0.001) c.p < 0.001; SOD: p < 0.001; CAT: p < 0.001) and 300 mg/kg treatment (GSH: 20.1 ± 2–42.3 ± 2.9 μg/mg protein (73%); SOD: 0.4 ± 0.1–1.1 ± 0.2 U/mg protein (58%); CAT: 11.6 ± 1.8–23.9 ± 2.1 U/mg protein (72%)) . Therefore, the results suggest that the LT treatment prevented VCR-induced decreases in sciatic antioxidation power.However, the decreased levels of GSH, SOD, and CAT in the V groups were significantly restored after the LT 100 mg/kg treatment (GSH: 20.1 ± 2.8–35.1 ± 2.7 μg/mg protein (49%); SOD: 0.4 ± 0.1–1 ± 0.2 U/mg protein (50%); CAT: 11.6 ± 1.8–20.3 ± 1.8 U/mg protein (51%))  ) (< 0.001)  in the rp < 0.001) and 300 mg/kg treatment (59.4 ± 7.9–35.1 ± 3.2 ppm/mg protein (−48%)) . These results indicate that the LT treatment inhibited VCR-induced increases in sciatic total calcium.The increased total calcium levels in the V groups were inhibited by the LT 100 mg/kg treatment (59.4 ± 7.9–42.9 ± 2.8 ppm/mg of protein (−33%))  (p < 0.001) (p < 0.001) (p < 0.001) (p < 0.001)  (< 0.001) a, IL-6 (< 0.001) b, MPO (1< 0.001) d, and ca< 0.001) e were in< 0.001) c was decp < 0.001; IL-6: p < 0.001; IL-10: p < 0.001; MPO: p < 0.001; caspase-3: p < 0.001) and 300 mg/kg treatment (TNF-α: 54.4 ± 9.1–32.3 ± 4.2 pg/mg protein (−51%); IL-6: 256.7 ± 9.3–156.1 ± 12.7 pg/mg protein (−55%); IL-10: 46.6 ± 7.8–129.9 ± 14 pg/mg protein (58%); MPO: 141.4 ± 12.8–70.4 ± 10.5 U/mg protein (−55%); caspase-3: 51.4 ± 2.6–30.9 ± 3.5 ng/mg protein (−49%)) . These results indicate that the LT treatment inhibited VCR-induced increases in sciatic neuroinflammatory activity, as well as apoptosis markers.The increases in TNF-α, IL-6, MPO, and caspase-3 levels and decrease in IL-10 levels in the V groups were inhibited by the LT100 mg/kg treatment (TNF-α: 54.4 ± 9.1–40.1 ± 3.9 pg/mg protein (−33%); IL-6: 256.7 ± 9.3–198.1 ± 12 pg/mg protein (−32%); IL-10: 46.6 ± 7.8–101.6 ± 10.8 pg/mg protein (39%); MPO: 141.4 ± 12.8–94.6 ± 11.6 U/mg protein (−36%); caspase-3: 51.4 ± 2.6–39.6 ± 3.5 ng/mg protein (−28%)) (V vs. V + T100, TNF-α: Based on this study, LT was found to protect against various VCR-induced pathologies, such as hyperalgesia, allodynia, oxidative damage, calcium dysregulation, neuroinflammation, and activation of the apoptotic pathway. This study was the first to demonstrate the potential of LT in mitigating VCR-induced pathophysiological dysfunction, possibly through antioxidative, calcium homeostasis, anti-inflammatory, and anti-apoptotic mechanisms, which could prove beneficial in treating chemotherapy-related peripheral neuropathy. Animal studies using the VCR-induced neuropathy model can be used to identify new treatments for chemotherapeutic-agent-associated neuropsychiatric disorders, as the behavioral alterations observed in rodents are similar to those seen in humans . On day Research indicated that VCR sensitizes dorsal horn nociceptive neurons, causing hyperalgesia and allodynia by inducing hyperresponsiveness in these neurons ,6,35. InOur findings indicate that calcium accumulation within cells increased neuronal oxidative stress in VCR-induced neuropathy . This bu2O2 and chloride ions, resulting in hypochlorite, which interacts with polyunsaturated fatty acids and induces lipid peroxidation [Following the LT therapy, the VCR-induced neuropathy was reduced, possibly due to its antioxidant properties, which restored animals’ nitrosative and oxidative stress indicators. MPO catalyzes the reaction between Hxidation . Neutropxidation . Inflammxidation . TNF-α, xidation ,46. Prevxidation . Treatmexidation ,49.Following the VCR injection, the levels of TNF-α, IL-1, IL-6, and MPO were found to be significantly elevated in the nerve tissue of rats, while the IL-10 levels were lower, which is consistent with prior research ,43. ThisExcessive nitrosative and oxidative stress can activate the inflammatory response and release inflammatory mediators that initiate the apoptotic pathway, which was suggested as a key mechanism for neuronal cell death and a critical pathway involved in the progression of neuropathy development ,5,6,7,8.It has long been believed that neuropathy is associated with abnormal neuronal activity in the sciatic nerve . LT’s anThe results of this study validate chemotherapy-induced neuropathy pharmacologically in a relatively broad sense. Nevertheless, more research is needed to determine its exact mechanism of action and full potential as an analgesic. The role of LT in treating clinically relevant human neuropathy and its benefits should be investigated in the future. In this study, the protective effects of LT might be explained by its antioxidant, calcium homeostasis, anti-inflammatory, anti-apoptotic, and neuroprotective properties .


Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor’s transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow. Fas/CD9 complex . Followi complex .Synthetic biology has become an alternative option to analyze cytokine signal transduction as well as for the development of personalized therapies . Recentlin silico prediction of structures of proteins and protein complexes   and squaomplexes . These domplexes  and Alphomplexes  will aidHere, we developed an experimental workflow for the Fas-SyCyR system to functionally characterize SNPs and structure-predicted mutations within the transmembrane and intracellular (death) domain of Fas/CD95. We used the SNP database (dbSNP) and clinical variants (ClinVar) database, compilations of all known polymorphisms and polymorphisms with a clinical correlation. Provean and structure-guided analysis were used for the preselection of candidate GOF and LOF mutations. Mutations were introduced into Fas-SyCyR, followed by a functional quantitative characterization including cellular proliferation, apoptosis, and effector caspases 3 and 7 cleavage assays.In summary, our results showed that among the 35 functionally characterized mutants, 22 were strong LOF, eight were mild LOF, and five led to a mild GOF. We comprehensively demonstrated that the Fas-SyCyR system is a valid tool to functionally and systematically characterize LOF and GOF variants.Among the 17,889 SNPs found in the Fas gene, we surveyed the listed 337 nonsynonymous missense SNPs of the transmembrane and intracellular domain, of which 39 were listed in ClinVar. From the 337 missense SNPs, 23 were reported in peer-reviewed publications, 19 had a direct disease association but only 13 were experimentally validated , 22.Nonsynonymous SNPs reported in ClinVar have a high probability of being LOF, whereas SNPs listed only in dbSNP might have no effect on protein function. To identify further nonsynonymous SNPs with potential LOF characteristics among the ones listed in dbSNP, we analyzed all nonsynonymous dbSNPs with Provean. We then selected ten previously uncharacterized nonsynonymous SNPs from ClinVar and five Provean predicted SNPs from dbSNP . Moreover, we included the five previously characterized nonsynonymous LOF SNPs Y232C, T241P, R250P, D260V, and T270I located in the intracellular DD, which were reported to be associated with ALPS and SCC , 30. FigVHHFas) receptor and introduced into Ba/F3-gp130 cells. Cell surface expression of mutant CVHHFas  was verified by flow cytometry using HA antibodies (A and B).The 20 selected nonsynonymous missense SNPs were introduced into the complementary DNA (cDNA) coding for the synthetic Fas-SyCyR  fused to an Fc part of an IgG1 antibody –induced proliferation of Ba/F3-gp130 cells expressing synthetic Fas as control (A and B). HIL-6 is a fusion protein of IL-6 and the soluble IL-6R, which specifically activates gp130 receptor signal transduction and proliferation of Ba/F3-gp130 cells . Among the 15 additional candidates, eight variants were also unable to inhibit HIL-6–induced cellular proliferation, thereof were six SNPs from ClinVar  and two SNPs predicted by Provean (G247R and D269H) (B).As expected, the five previously characterized LOF SNPs  failed to inhibit HIL-6–induced proliferation A. Among d D269H) B.50 of 31 ng/ml and 29.8 ng/ml, respectively. The remaining SNPs K251T, T319I, S320G did not change the biological activity of Fas . Of note, the previously uncharacterized SNPs I233V and D321N were mild GOF mutations with an IC50 of 0.11 ng/ml and 0.20 ng/ml, respectively  and three mutants from the dbSNP/Provean prediction  were biologically active. The two variants S230R, L315F showed, however, a reduced ICectively .Figure 4Here, cellular proliferation was used to initially estimate the consequences for the biological activity of the Fas SNP mutations. In summary, 13 out of 20 SNPs are complete LOFs , two have mild LOFs , three have no effect , and two are GOFs .Using structure-based prediction, we selected 15 LOF and GOF candidates. All 15 missense mutations were located in the DD and resulted in substitution into an alanine  or an alternative amino acid  to achieve maximal repulsion between the DD and FADD. In detail, D228S causes disruption of a salt bridge with FADD R142, while Y291D leads to loss of hydrophobic interaction with L172, L176, V173, and N107. VHHFas. Ba/F3-gp130 cells expressing the CVHHFas variants were generated, and cell surface expression was verified by flow cytometry (VHHFas (variant) cells were stimulated with 3C and HIL-6. The CVHHFas alanine variants L224A, L229A, K231A, T235A, I236A, G238A, L298A and the two additional variants D228S, Y291D failed to inhibit HIL-6–induced proliferation, suggesting that these mutations cause LOF (A). On the other hand, N223A, I295A, L306A and N302D, Q311E, N329D still prevented cellular proliferation. Of these, I295A and L306A showed reduced inhibitory activity considering that higher concentrations of 3C were needed to abrogate cellular proliferation (IC50 = 9.49 ng/ml and not to be determined). Moreover, N223A and N329D were more effective (IC50 = 0.35 ng/ml and IC50= 0.10 ng/ml) and might represent mild GOF mutations (B).The 15 structure-based mutations were introduced into the cDNA coding for Cytometry . Ba/F3-gause LOF A. On theutations B.Figure In summary, 9 out of 15 SNPs are complete LOFs , two are mild LOFs (I295A and L306A), two indicated no effect , and two are proved to be GOFs (N223A and N329D).VHHFas variants after 24 h stimulation with HIL-6 alone or in combination with the synthetic Fas ligand 3C. As illustrated in the schematic plots (A), the addition of HIL-6 resulted in 87.7% living Ba/F3-gp130-CVHHFas cells, whereas co-incubation with 3C resulted in 74% apoptotic cells. The previously characterized SNP variants Y232C, T241P, R250P, D260V, T270I failed to induce apoptosis  or with no effect  in the proliferation assays efficiently produced late apoptotic cells . Of note, the remaining three variants , L315F) displayed fewer late apoptotic cells , from which S230R and L315F were also seen in the proliferation assay to be mild LOF variants . Likewise, the eight other identified LOF SNPs from ClinVar and Provean C178R, G247R, G253V, I262N, D269H, E272G, E289D, and N326H (B) failed to induce caspase 3/7. Eight out of the nine others identified structure-based LOF mutations L224A, L229A, K231A, T235A, I236A, G238A, Y291D, and L298A (C) were unable to activate the effector caspases, although D228S still resulted in about 20% activation (C). D228S causes the loss of a salt bridge interaction between Fas and FADD. In the D228S, the serine is still within 4.5 Å distance to R142 of FADD and may hence be able to form a H-bond which does not completely abrogate the interaction but significantly weaken it.An early hallmark of apoptosis is the activation of the effector caspases 3 and 7 , which wtivation A. Likewind N326H B failed nd L298A C were untivation C. D228S VHHFas, induction of caspase 3/7 of the active SNPs S230R, I233V, K251T, L315F, T319I, S320G, D321N was 37%, 134%, 64%, 52.5%, 135%, 39% and 28%, respectively (A).Compared to Cectively A.Figure B). In detail, the effector caspase activation of N223A and N329D mutants supported the previous proliferation and late apoptosis data as them as novel GOF mutants, with a respective percentage equal to 234% and 131% compared to CVHHFas, respectively. On the contrary, I295A, N302D, L306A, and Q311E induced 32%, 36%, 17%, and 40%, respectively, resulting in mild LOF mutations.Similarly, the structure-based mutations N223A, I295A, N302D, L306A, Q311E, and N329D induced caspase 3/7 as well, albeit with different efficiencies compared to stimulation of cells expressing the WT receptor B. In detWe have summarized all findings in One plausible application for SyCyRs could be to strengthen chimeric antigen receptor (CAR) T-cell therapies, either supporting or suppressing the activity of engineered T-cells. Following this aim, we were curious to test if combining GOF or LOF mutations would result in stronger and weaker activation of synthetic WT Fas.A), did not induce late apoptosis .The SNP L315F and the alanine mutants I295A and L306A had consistently reduced Fas activity in our assays. Therefore, we combined them in a triple mutant to analyze if this will result in a more severe LOF phenotype. As expected, the combination of the mild LOF L315F, I295A, and L306A in one variant resulted in complete LOF, since this variant was unable to inhibit the proliferation of Ba/F3-gp130 cells A, did no50 = 19.01 ng/ml) (D), to induce apoptosis (38% of late apoptotic cells versus 98% for WT Fas) .On the other hand, the mutants N223A and N329D alone conferred a stronger capacity to inhibit proliferation, partially reduced amounts of late apoptotic cells, and significantly higher caspase 3/7 activity compared to WT, after activation with synthetic Fas. Hence, likewise, we combined those two mutants to analyze if this will result in a stronger GOF phenotype. Unexpectedly, the combination of two stronger activated mutations N223A and N329D resulted in a partial LOF variant with a lower capacity to inhibit proliferation (IC1 ng/ml) D, to indIn conclusion, the combination of three less active mutations  resulted in a complete LOF phenotype, while the combination of two GOF mutations (N223A plus N329D) did not lead to a more active variant.VHHFas WT and mutant cells with an additional synthetic GVHHFas cDNA (A). Cell surface expression of GVHHFas was verified by flow cytometry using Myc-tag antibodies (A). We tested the two previously described LOF mutations T241P and D260. As shown in Figure 12B , which were LOF in our characterization. Interestingly, those mutations still impaired apoptosis compared to normal Fas but with a percentage of apoptotic cells equal to 78%, 49%, and 26%, respectively  GOF mutations associated with Fas, with or without a direct immune diseases correlation. We initially predicted relevant SNPs found in the transmembrane and intracellular human Fas domain by in-depth structure-guided analysis  or by baet al. showed how two ALPS-affected siblings were carrying the same mutation Y232C in Fas gene causing a defect in apoptosis  and −86.3 KJ/mol (N329) in the open conformation (PDB: 3EZQ)  and −32.1 KJ/mol (N329) in the closed conformation  with 10% fetal calf serum , 60 mg/l penicillin, and 100 mg/l streptomycin (Genaxxon Bioscience GmbH), and supplemented with Hyper-IL-6, a fusion protein of IL-6 and soluble IL-6 receptor  (FlowJo LLC).α-HA-tag mAb  was used to identify the expression of Fas-SyCyR  in the transfected Ba/F3-gp130 cells. The cells were resuspended in 50 μl of fluorescence-activated cell sorting (FACS) solution plus primary antibody HA after being washed in FACS buffer . Cells were washed and resuspended in 50 μl FACS buffer with secondary antibody Alexa Fluor 488–conjugated Fab goat anti-rabbit IgG  before being incubated for at least 1 h at room temperature. Cells were washed, resuspended in 200 μl of FACS buffer, and the fluorescence signal was acquired by flow cytometry . Data were analyzed with FlowJo V10 . As previously described (VHHFas WT cells stimulated with lowest concentration of 3xmCherry-Fc (1 × 10−4 ng/ml) was set as 100%, and the efficiency of the other CVHHFas variants was calculated accordingly.Ba/F3-gp130 cells were washed three times with PBS. 5 × 105 cells were seeded and stimulated for 24 h with HIL-6 (20 ng/ml) with and without 3xmCherry-Fc (100 ng/ml). Afterward, cells were washed twice with ice-cold PBS and resuspended in 50 μl Annexin-V–binding buffer (BD Bioscience) and Annexin-V (1:600) (ImmunoTools). Following incubation at room temperature for 15 min in the dark, 1 μl 7-AAD (R&D Systems) was added and diluted in 200 μl of Annexin-V–binding buffer. Analysis was carried out by flow cytometry . Data were evaluated using FlowJo V10 (FlowJo LLC).Ba/F3-gp130 cell lines were washed three times with PBS. 2.5 × 105 cells were seeded in 100 μl/well in 96 wells/plate and stimulated with 20 ng/ml HIL-6 with and without 100 ng/ml 3xmCherry-Fc. After 6 h stimulation, 1 μl of 1 mM caspase 3/7 inhibitor Ac-DEVD-CHO stock solution was added only in the selected samples for 10 min at room temperature, and subsequently, 100 μl of caspase 3/7 working solution was added for at least 1 h at room temperature. Fluorescence intensity was measured at Ex/Em= 490/525 nm. For each condition of an experiment, three wells were measured. All values were normalized by subtracting background values from the final measurement. Caspase 3/7 activation after addition of 3xmCherry-Fc in Ba/F3-gp130 cells expressing CVHHFas was set to 100%, and the efficiency of the other CVHHFas variants was calculated accordingly.Effector caspases 3 and 7 activation was detected using Amplite Fluorimetric Caspase 3/7 Assay Kit ∗Green Fluorescence∗ . Briefly, Ba/F3-gp130 cell lines were washed three times with PBS. 1 × 10p < 0.05 .Data are shown as mean ± SEM. Multiple comparisons were determined with GraphPad Prism 6 (GraphPad Software) using one-way ANOVA column analyses. Statistical significance was set to 3EZQ.Structural analysis of FAS and FADD was performed using UCSF Chimera  based onjscheller@uni-duesseldorf.de).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jürgen Scheller (This study did not generate new unique reagents. All cDNAs are available upon request.in vivo and to analyze the potential combination with the CAR T-cell treatment.As shown and discussed before, this study shows early research upon the usage of a new synthetic cytokine system. Further studies are necessary to verify these findings This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Limited studies explored the relationship between lymphocyte recovery after definitive concurrent chemoradiotherapy (dCCRT) and prognosis in esophageal squamous cell carcinoma (ESCC).ESCC patients with obtainable absolute lymphocyte counts (ALCs) at 6 months after dCCRT were screened from prospective trials. Patients were divided into groups according to the grade of ALC nadir during radiotherapy (G4 or G1–3) and lymphocyte recovery status, which was assessed by lymphocyte recovery index (LRI), calculated as the ratio of post- to pre-treatment lymphocyte counts. Cox analysis was conducted to evaluate the prognostic significance of lymphocyte recovery status. Irradiated relative volumes of the bone marrow (BM) and spleen and effective dose to immune cells (EDIC) were collected to identify their impacts on lymphocyte recovery status by logistic analysis.232 patients were enrolled. In 69 patients with G4 ALC nadir (group A and B) and 163 patients with G1–3 ALC nadir (group C and D) during dCCRT, 27 (group A) and 67 (group C) patients showed an insufficient level of lymphocyte recovery (LRI < 60%), and 42 (group B) and 96 (group D) patients showed a satisfactory level of lymphocyte recovery (LRI ≥ 60%). Cox multivariable analysis revealed that inadequate lymphocyte recovery was significantly associated with worse overall survival  and local recurrence-free survival  both in group A vs group B and group C vs group D. Logistic analysis identified BM V5 (OR 4.24 and 2.29) as an independent predictor of inadequate lymphocyte recovery from G4 or G1–3 ALC nadir, respectively.Insufficient lymphocyte recovery might serve as a valuable prognostic factor, regardless of whether patients experienced G4 or G1–3 ALC nadir during radiotherapy. Additionally, it was observed that a larger relative volume of BM receiving ≥ 5 Gy was correlated with a higher risk of insufficient lymphocyte recovery.The online version contains supplementary material available at 10.1186/s13014-023-02354-w. Radiotherapy as a recognized anti-tumor approach, is a double-edged sword as it also leads to the depletion of immune cells , 2. LympThe recovery of lymphocyte counts after radiotherapy is a time-consuming process . A studyBuilding upon previous research findings, this study aimed to explore the relationship between lymphocyte recovery from radiation-induced lymphopenia and survival outcomes in patients with esophageal squamous cell carcinoma (ESCC) who underwent definitive concurrent chemoradiotherapy (dCCRT), and further to figure out which specific lymphocyte-related organ at risk exhibit a stronger correlation with lymphocyte recovery.NCT01591135) [NCT02459457) [ESCC patients treated with dCCRT were screened from two prospective randomized clinical trials known as ESO Shanghai 1 1591135)  and ESO 1135  and2459457) , we next2459457) , 9.The patients were followed up every 3 months for the first two years after the whole treatment and then every 6 months to the fifth year, when the follow-up time could be prolonged to every year in no exceptional circumstances. The follow-up duration lasted for at least 6 years. Overall survival (OS), progression-free survival (PFS), local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) were recorded , 9.9/L), lymphopenia is defined as ALC < 1.1 × 109/L; the degree of lymphopenia is divided into 0–4 levels as follows: 0 (≥ 1.1 × 109/L); 1 (< 1.1–0.8 × 109/L), 2 (< 0.8–0.5 × 109/L), 3 (< 0.5–0.2 × 109/L) and 4 (< 0.2 × 109/L). The lowest ALCs during dCCRT were identified by G0–4. The status of lymphocyte recovery was estimated by lymphocyte recovery index (LRI), which was defined as the ratio of ALC at 6 months after the end of chemoradiotherapy and ALC at baseline.Absolute lymphocyte counts (ALCs) were collected before dCCRT (at baseline), each week during dCCRT, and 6 (± 1) months after dCCRT. Based on CTCAE version 5.0 and the lower normal limit of ALC in our hospital  were regarded as “unrecovered”, while those with adequate lymphocyte recovery (LRI ≥ cut-off) were regarded as “recovered”. Then based on the grade of lymphocyte nadir during dCCRT (G4 and G1–3), the population were categorized into 4 groups: (1) Group A (G4 → unrecovered); (2) Group B (G4 → recovered); (3) Group C (G1–3 → unrecovered); (4) Group D (G1–3 → recovered).The body, heart, lungs, and spleen were outlined according to RTOG 1106 Atlas. The mean dose of heart, lungs and body were combined to calculate the effective dose to immune cells (EDIC) developed by Jin et al. [p-value of each variable was calculated in the Cox model. Dose-volume parameters predicting lymphocyte recovery were transformed into binary variables by receiver operating characteristic curve (ROC) analysis and the organ-specific parameter with the lowest p value on univariate was chosen into multivariable adjustment. Univariable and multivariable logistic regression analysis were performed to correlate lymphocyte recovery with dose-volume parameters. Variables with p < 0.1 on univariable Cox/Logistic analysis were input for the following multivariable analysis. A two-tailed p < 0.05 was considered statistical significance. R studio version 4.2.1  and Graphpad Prism version 9.0  were applied in this study.Continuous variables in clinical characteristics were categorized by median splits. The optimal cut-off point of LRI taken the integer portion was determined by the maximally selected log-rank statistics based on OS using the R package “maxstat” . Kaplan Two hundred thirty-two eligible patients were enrolled for this study, including 185 (79.7%) males and 47 (20.3%) females. The median age of all patients was 62 years. Most of them (69.8%) were in normal health conditions with ECOG-PS-0. Fifty-eight (25.0%) patients at stage II and 137 (59.1%) patients with tumors at cervical and upper esophagus. The median tumor length was 5.0 cm. Of 232 patients, 94.8% finished the 61.2 Gy radiotherapy course. The number of patients treated with PF, TF, TP and TC was 64 (27.6%), 102 (44.0%), 30 (12.9%) and 36 (15.5%), respectively. After concurrent chemoradiotherapy, 178 (76.7%) patients received 2 cycles of consolidation chemotherapy and 54 (23.2%) patients received 0–1 cycle. Clinical characteristics in details were listed in Table 9/L) 1.68, and 0.99, 0.80, 0.61, 0.50, 0.41, 0.43, 0.43 for week1–7 during dCCRT, respectively. The cumulative incidence of G4 ALC nadir and G1–3 nadir was 29.7% (N = 69), and 70.3% (N = 163).The ALC data of 232 patients were obtained. During treatment, ALCs declined every week and generally reached a plateau at week 5, continuing till the end of treatment, then gradually elevated to near-normal levels Fig. . The med9/L), while more than half of them (55.2%) were still accompanied by varying degrees of lymphopenia: 77 (33.2%) with grade 1 lymphopenia, 47 (20.3%) with grade 2 and 4 (1.7%) with grade 3. The median of ALC at 6 months after dCCRT was 1.06 × 109/L (range 0.41–3.98).Six months after the end of dCCRT, there were 104 (44.8%) patients whose ALCs returned to normal level (≥ 1.1 × 10With regard to the status of lymphocyte recovery the median of LRI was 67.8% range 19.8–193.1). According to the cut-off point of LRI (60%) by maxstat based on OS  and 5-year PFS rate  were observed in group A vs group B. Also, prominent differences in the 5-year LRFS rate  and 5-year DMFS rate  existed in group A vs group B. Among patients with G1–3 ALC nadir during dCCRT, survival curve analysis indicated poorer 5-year OS  and 5-year PFS  in group C vs group D. The 5-year LRFS rate  and 5-year DMFS rate  in group D was respectively significantly higher than that in group C.At analysis, 117 (50.4%) patients died with 69.9 months of median OS time. The overall PFS, LRFS and DMFS median time was 27.4 months, 46.2 months and 49.2 months, respectively. As displayed in Fig. p = 0.005) and 5-year PFS rate 3.7% vs 38.0% (p < 0.001), respectively. Besides, poorer 5-year LRFS rate  and 5-year DMFS rate  were observed in group A vs group C.Comparing group A with group C, OS and PFS were both worse in patients with G4 ALC nadir than those with G1–3 ALC nadir, with a 5-year OS rate 18.5% vs 46.8% months (p = 0.002) and PFS , as well as poorer LRFS  and DMFS . Among patients with G1–3 ALC nadir during dCCRT, inadequate lymphocyte recovery was still found to be an independent factor associated with poorer OS  and LRFS . However, no statistically prognostic significance was observed in PFS and DMFS , shorter PFS , as well as inferior LRFS  and inferior DMFS .Based on the tumor stage, the population was re-stratified for further investigation. In the subset of patients with stage II (N = 58), no significant correlations were observed between lymphocyte recovery status and clinical outcomes (data not shown). However, within the stage III + IV group , patients who did not experience sufficient lymphocyte recovery at 6 months post-therapy had inferior OS, PFS, LRFS and DMFS , 33.5% (range 9.3–54.2), 26.1% (range 5.5–41.8), 18.8% (range 3.3–34.7), 5.3% (range 0.5–16.2), respectively. The max of the spleen V5, V10, V20, V30 and V50 was 96.0%, 87.7%, 80.3%, 55.2% and 6.7%, respectively. For EDIC, the median dose was 9.0 Gy (range 2.0–14.6).In group A vs B, the median of V5, V10, V20, V30 and V50 of the BM was 46.0% vs 40.7%, 37.2% vs 33.8%, 27.9% vs 25.7%, 20.3% vs 19.9% and 5.3% vs 5.4%, and the max values of spleen V5, V10, V20, V30 and V50 was 77.4% vs 96.0%, 60.0% vs 87.7%, 35.5% vs 80.3%, 7.9% vs 55.2% and 0 vs 1.4%, respectively. In group C vs D, the median of related BM dose-volume parameters mentioned above was respectively 44.7% vs 40.8%, 34.0% vs 32.2%, 26.3% vs 25.5%, 19.0% vs 17.6% and 5.5% vs 4.8%; and max values of spleen V5, V10, V20, V30 and V50 were 70.0% vs 87.0%, 60.5% vs 64.7%, 50.7% vs 53.8%, 32.4% vs 45.8% and 6.5% vs 6.7%. The median of EDIC in group A vs B and group C vs D was 14.1 Gy vs 13.6 Gy and 8.59 Gy vs 8.50 Gy, respectively.p-values of less than 0.05 whether in comparison with group A and group B  was positively associated with lymphocyte unrecovery, while, BM V5 ≥ 46.0%  for patients with G1–3 ALC nadir and lymphocyte unrecovery. Subsequently, multivariable adjustment indicated that BM V5 ≥ 40.7%  and ≥ 46.0%  was independently correlated with lymphocyte unrecovery 6 months after dCCRT respectively in patients with G4 and G1–3 ALC nadir , dose volume parameters of BM gave To the best of our knowledge, this study represents the first exploration of lymphocyte recovery at 6 months after dCCRT in ESCC patients. We employed a novel index, integrating lymphocyte counts at the post-6-month mark with baseline counts, to demonstrate the status of lymphocyte recovery. The findings revealed that patients with inadequate lymphocyte recovery had poorer survival outcomes, simultaneously indicating the potential of this recovery index to differentiate prognosis in patients with G1–3 ALC nadir during dCCRT, which was previously overlooked. Furthermore, a multivariable analysis revealed a significant correlation between bone marrow irradiation and lymphocyte recovery.It is widely acknowledged that circulating lymphocytes serve as promising markers for evaluating the systemic immune system .Notably,Notably, Lee, et al. , found tThe weak correlations between lymphocyte recovery and other organs at risk might be attributed to the fact that blood vessels and the spleen serve as lymphocytic reserve organs rather than hemopoietic organs. Irradiation to circulating cells and the spleen was more likely to cause acute elimination of lymphocytes during radiotherapy instead of affecting lymphopoiesis. What cannot be ignored is that we used a surrogate to represent the irradiation of various organs with large vessels, and due to different tumor locations, not every spleen or bone marrow of each patient was covered within the same scanning range. On the other hand, the bone marrow in this study was delineated by the external contours rather than the low-density regions within the bones.There are some limitations in our study. First, it is a retrospective study, meaning that we could hardly control other factors that may affect ALCs, such as different chemotherapy regimens. Second, flow cytometry was not applied to identify the lymphocyte diversity, which plays a prominent part in immune response. Third, this preliminary finding should be verified in a larger population. Fourth, the relative volumes of organs at risk utilized in this study may be susceptible to variation depending on factors such as the scan range and patient positioning. Fifth, newer edition for evaluating tumor stage should be applied in future research. Last, it's worth investigating which part of bone marrow takes more responsibility to lymphocyte recovery in esophageal cancer, or evaluating longitudinal changes in hematopoietic function of bone marrow by PET.Despite these aforementioned limitations, our study yielded valuable insights into the association between an insufficient level of lymphocyte recovery after dCCRT and poorer survival outcomes in patients with ESCC, regardless of the severity of ALC nadir (G4 or G1–3) during radiotherapy. What’s more, our findings highlighted the potential impact of bone marrow irradiation on lymphocyte recovery, emphasizing the need for strict restrictions on nonessential bone marrow irradiation during radiotherapy..Inadequate lymphocyte recovery at 6 months after dCCRT for ESCC was an independent prognostic indicator of unfavorable survival outcomes, regardless of whether it occurred in patients who experienced G4 or G1–3 ALC nadir during radiotherapy. Additionally, there was a significant association between bone marrow irradiation and lymphocyte recovery.Additional file 1: Supplementary Figures 1–3.Additional file 2: Supplementary Tables 1–8.