Finally, metabolic characterizations of the tagged DAGs were further explored in L-Arginine-induced acute pancreatitis mice and resveratrol treated model mice. The results indicated that 1,2-DAGs were increased in the serum of model mice relative to normal controls and resveratrol significantly altered this metabolic abnormality. The currently established DMG/DMA-oriented paired charge derivatization strategy is promising for depicting DAGs changes more accurately in metabolic studies of lipid-related diseases and accurately evaluating drug treatment strategies.Diacylglycerols (DAGs) are important lipid mediators in cellular signaling transduction and metabolism. Imbalanced production or consumption of DAGs has a negative impact on the physiological functions of the body. However, comprehensive monitoring of structurally diverse DAGs remains a daunting task due to the rapid metabolism and ion suppression characteristics in biofluids. These bottlenecks call for developing a method that enables sensitive quantification of DAGs in biological sample. In this work, a straightforward charge derivatization strategy was developed to insert a series of structure analogs charge label, i.e., N, N-dimethylglycine (DMG) and N, N-dimethylalanine (DMA), on the free hydroxyl group of the DAGs. Owing to the existence of tertiary amino groups in charge label, the mass spectrometry ionization response of the derivatized DAGs was significantly increased in comparison with traditional metal ion adducts. After charge derivatization, the specific neutral loss diagnostic ions  were captured by mass spectrometry. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the characteristic diagnostic ions of the derivatized DAGs have been developed as sensitive methods for the detection (detection limit = 16 aM) and quantification (quantification limit = 62.5 aM) of DAGs in serum. Moreover, the tagged 1,2-DAGs and 1,3-DAGs  Neutral lipids participate in numerous metabolic pathways and play a key role in cellular energy supply, lipid synthesis and cellular signaling . As an ivia ester bond  selectively reacted with DAGs in mild condition and rapidly generated positive ion-specific tag fragment during ESI-MS electrospray ionization. DMA, as a charge-tag for DMG methylation, was used as one-to-one internal standards to correct for variations in the analysis. To our acknowledge, the DMG/DMA-oriented paired charge derivatization method was first proposed. In this study, synthetic DAGs standards with variable chain length and position location were utilized to optimize reaction condition and establish a quantitative MS/MS approach. This is an integrated approach that allows for parallel measurement of multiple chain lengths and positional heterogeneity of DAGs in a single run. The analytical capability of the proposed strategy was validated by monitoring DAGs in serum sample of acute pancreatitis and profiling variations of DAGs after resveratrol intervention. In light of the biological importance of DAGs in acute inflammation and numerous other pathological conditions, the presented DMG/DMA-oriented charge derivatization method is advisable for tracking the alterations of DAGs in pathological state and accurately evaluating drug treatment strategies.All the chemical reagents and solvents were obtained from commercial sources and were utilized without further purification. N, N-dimethylglycine (DMG), N, N-dimethylalanine (DMA), N-(dimethylamino) pyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich . DAG 18:0/18:0/0, DAG 16:0/18:1/0, DAGs 16:0/16:0/0, DAG 14:0/14:0/0, DAG 12:0/12:0/0, DAG 10:0/10:0/0 and DAG 8:0/8:0/0 were purchased from Avanti PolarLipids . DAG 18:0/0/18:0, DAG 16:0/0/18:1, DAG 18:1/18:1/0, DAG 18:1/0/18:1 were purchased from Nu-Check Prep, Inc. . Chromatographic grade methanol and acetonitrile were purchased from Merck KGaA . Mass spectrometry grade formic acid and other chemical reagents were obtained from Sigma-Aldrich Laboratories, Inc.n = 40) were randomly divided into different research groups (n = 10). First group is blank control group, in which mice were administrated with the saline . The second group was the acute pancreatitis model group, in which mice were treated with two intraperitoneal injections of L-arginine . The third group was the low-dose resveratrol  combined with L-arginine group, in which mice were given the previously described L-arginine treatment and were administered resveratrol twice daily for 3 days. The fourth group was the high-dose resveratrol  combined with L-arginine group, in which mice were treated with both resveratrol and L-arginine as described above. After injecting L-arginine for 1 h, resveratrol was administered by gavage twice daily for three consecutive days. L-arginine was prepared by solubilizing it in 0.9% saline, the pH of this solution was adjusted to 7 with hydrochloric acid. After animal treatment, serum samples were collected from above four groups. Simultaneously, lungs were taken. All samples were stored in a −80°C refrigerator.Male healthy C57BL/6 mice were provided by China Pharmaceutical University . All mice were housed in an air-conditioned rearing chamber with an ambient temperature of 25 ± 2°C, relative air humidity of 50% ± 10% and the cycle of light and darkness is 12 h. Water and food were allowed to be freely accessible during the entire study. Animal studies have been approved by the Animal Ethics Committee of China Pharmaceutical University. After 1 week of rearing acclimatization, mice . Subsequently, all reagents were respectively added into glass tube and then vortex for 1 min. After centrifuging the tubes at 2,500 rpm for 1 min, the sample in glass vessels reacts at 45°C for 60 min. The derivatization reaction was quenched by introducing 1.5 ml CH2Cl2/MeOH  mixture and 0.5 ml NH4OH (25 mM) into the glass tubes. After vortexing the glass tubes for 1 min, the sample was allowed to stand for 5 min. Next, collect the lower CH2Cl2 layer and blow dry with nitrogen. A modified Bligh-Dyer method was applied for extraction and desalination of the derived DAGs. Finally, 75 μl mobile phase (50% Isopropanol-50% Acetonitrile) was utilized to re-suspend extractive product for MS analysis. The above charge derivatization procedure was practiced to directly modify DAGs in biological sample.DAGs (0.2–200 nM), DMG (0.125 mM), DMAP (0.5 mM) and EDC (0.25 mM), were dissolved in acetonitrile and dichloromethane  and 10 μl of serum were introduced into a glass tube and the mixture was swirled for 2 min. Subsequently, 100 μl of deionized water was introduced and then the CH2Cl2 layer was collected. An amount of CH2Cl2 extract was shifted to a glass vial and the sample was evaporated until dry under N2 airflow. All dried samples were derivatized according to the derivatization principle described above.For the preparation of biological samples, liquid-liquid extraction was performed to remove proteins and concentrate lipids. At first, 400 μl of ice-cold CHAn AB Sciex Triple Time-of-Flight 5,600 system in positive ion electrospray ionization was utilized for characterization of tagged DAGs. The quantification of DAGs derivatives was performed on Qsight LX50 UHPLC together with a Qsight 220 MS system . The temperature of the UHPLC autosampler was maintained at 4°C and the sample injection volume was adjusted to 2 μl. Chromatography separation was performed on a Waters Acquity BEH C18 column  at 55°C. The mobile phase consisted of solvent A (40% water-60% MeOH contains 5 mM ammonium acetate) and solvent B (90% isopropanol-10% acetonitrile with 0.1% formic acid). The mobile phase was delivered at a flow rate of 0.2 ml/min. The gradient program used was as follows: 10% B in 0–2 min, 10% B to 30% in 2–5 min, 30% B to 35% in 5–8 min, 35% B to 37% in 8–10 min, 37% B to 37% in 10–12 min, 38% B to 38% in 12–14 min, 38% B to 45% in 14–18 min, 45% B to 65% in 18–20 min, 65% B to 80% in 20–25 min, 80% B to 95% in 25–26 min, 95% B in 26–27 min, 10% B in 27–30 min. The operating parameters of the mass spectrometer were setup as below: dry gas value is 100, atomized gas value is 180, electrospray voltage is 5,500 V, and source temperature is 400°C.Unlike polar lipid, DAGs possess relatively lower ionization efficiency under ESI-MS, because there is no fixed ionizable group in the structure. Hence, it is advisable to insert a fixed charge-tag to DAGs for enhancing ESI ionization efficiency. Three commercially available N-methylcarboxylic acid derivative reagents, comprising DMG, DMA and N, N, N-trimethylglycine (TMG), were utilized to establish the derivatization procedure (structures were shown in +), m/z 694.5979 ([DAG + DMA]+), m/z 694.5986 ([DAG + TMG]+) in positive ion mode. Furthermore, all charge labeling have greatly enhanced the ionization of DAG in ESI-MS. In the case of DMG, the ionic intensity of the derived DAGs was superior to the formation of sodium adducts  failed to produce a product  and temperatures  were tested to obtain optimal derivatization. The conditions were selected to be examined in order to keep the solvent system under boiling point and avoid using a reflux condenser, thereby ultimately obtaining a better reaction rate. Based on the MS intensities achieved from the labeled DAGs standards, the intensities of the DAGs at 60 min were higher than those at 90 min and 120 min . On thisDMG-DAGs and DMA-DAGs were shown in DMG-DAG 18:0/18:0/0, m/z 724→m/z 607 for DMA-DAG 18:0/18:0/0, m/z 680→m/z 577 for DMG-DAG 16:0/18:1/0, m/z 694→m/z 577.1 for DMA-DAG 16:0/18:1/0, m/z 654→m/z 551 for DMG-DAG 16:0/16:0/0, m/z 668→m/z 551 for DMA-DAG 16:0/16:0/0, m/z706→m/z 603 for DMG-DAG18:0/18:0/0, m/z720→m/z 603 for DMA-DAG 18:0/18:0/0. The chromatograms of DAGs in UPLC-MS analysis were showed in DMG-DAGs and DMA-DAGs possess stable and reproducible characteristic neutral loss fragment.The properties of the 103 Da tag NLS were evaluated in terms of quantification of DAGs under optimal reaction conditions. The mass spectrometry quantitative parameters were optimized in sn-1,2 DAGs from the sn-1,3 DAGs. A gradient elution procedure of mobile phase was performed to separate different isomers of DAGs . The mixture was swirled for 2 min at 4°C and H2O (100 μl) was added. The obtained solvent was homogenized and then allowed to stand for 5 min. Finally, the upper layer of organic phase (250 μl) was collected and dried by evaporation to perform derivatization.Calibration standards, quality control (QC) samples and surrogate matrices excluding DAGs were prepared for the validation of MS quantification method. A series of standard solutions at ten levels of concentration  were prepared by dilution of DAG stock solutions with dichloromethane. QC samples were obtained by mixing each biological extract in equal volumes. A modified Bligh and Dyer procedure was applied for preparing surrogate matrices without DAGs. At first, 10 μl of serum was shifted into a 10 ml glass pipe, followed by the addition of 400 μl of cold CHr2). The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) in MS detection were used to assess the sensitivity of this method. The LLOQ should satisfy a signal-to-noise ratio (S/N) greater than 10 and a relative standard deviation (RSD) of the assay results less than or equal to 20% (n = 3). The lower limit of detection (LLOD) requires a signal-to-noise ratio greater than three and an RSD less than or equal to 20% (n = 3).Linearity and sensitivity: Solvent calibration curve and surrogate matrix calibration curve were established by adding calibration standards in pure solvent and surrogate matrices without DAGs in . LineariThe availability of alternative matrix curves was evaluated by comparing the slope of the alternative matrix curve and the solvent curve. The concentrations of QCs samples were calculated from both types of curves and the deviations between the profiles were evaluated. After parallelism was confirmed, three standard solutions at low, medium and high concentrations were selected for subsequent validation analysis based on the linear range of the alternative matrix curves.The matrix effect (ME) is measured as the ratio of three concentrations of standards (neat solution samples) to standards added to the alternative matrix (matrix samples). The ME was calculated by the following equation, where R represents the detection response of the DAGs.n = 3, three copies of each concentration were prepared). Recovery (%) = (A/B) × 100%. Matrix effect and recovery were listed in Extraction recovery efficiency was measured from the concentration of standards before (A) and after extraction (B), where the concentration was calculated from the alternative matrix curve (n = 6). The storage stability of the autosampler (4°C) for the derivatized DAG was inspected by testing QC samples over a 48-h period. In addition, the stability of IS at 4°C for 48 h was also assessed. As shown in Repeatability and stability were examined by analyzing six replicates of the QC samples in parallel. Precision was investigated by intra-day and inter-day RSD, which were measured in 1 day and on three consecutive days . The changes in the histological structure of the pancreas and lungs from the acute pancreatitis model animals were depicted by H&E staining . No altesn-isomers, including DAG 18:0/18:0/0, DAG 18:0/0/18:0, DAG 16:0/18:1/0, DAG 16:0/0/18:1, DAG 18:1/18:1/0, and DAG 18:1/0/18:1 were separately detected. The intensity changes of DAGs from four sets of samples were summarized in N = 6). The content of DAG 18:0/18:0/0, DAG 18:0/0/18:0, DAG 16:0/16:0/0, and DAG 16:0/18:1/0 increased by 3.4 ± 2.1, 3.5 ± 2.0, 3.6 ± 1.5, and 2.3 ± 1.8 times in models as compared to the control, respectively. After resveratrol intervention, the levels of DAGs, including sn-1,2 and sn-1,3-DAGs, both decreased. To investigate the effect of resveratrol on positional isomers of DAGs, the ratio of sn-1,2-DAGs to sn-1,3-DAGs in different groups was analyzed. sn-1,2-DAGs to sn-1,3-DAGs in four groups. Sn-1,2-DAGs, including DAG 16:0/18:1/0, DAG 18:0/18:0/0, and DAG 18:1/18:1/0, showed an elevated trend in the pancreatitis model. In the resveratrol treatment group, the ratios of sn-1,2-DAGs to sn-1,3-DAGs was decreased. This data suggests that resveratrol may have more influence on sn-1,2-DAGs production when regulating lipolysis. Previous studies have also revealed that resveratrol may regulate certain enzymes involved in the production of DAGs, such as phospholipase C or adipose triglyceride lipase (Additionally, we were interested in checking whether the DMG label and 103 Da NLS were available for the analysis of DAGs in the serum of mice with acute pancreatitis. e lipase . In futusn-isomers have been well-separated on the reversed-phase column. The proposed method was further adapted to the DAGs profiling of mice serum. Analysis of serum samples from mice with acute pancreatitis demonstrates that DAGs are found with significant changes of sn-1,2-DAGs in model plasma relative to normal control. Thus, the potential of the proposed the DMG/DMA-oriented paired charge derivatization method for detecting and quantifying DAGs selectively is expected to play an important role in metabolic studies of diseases associated with DAGs.In this study, we have successfully established a strategy by pairing charge derivatization with UHPLC-MS/MS for the sensitive quantification of DAGs in biological samples. The free hydroxyl groups of DAGs were derivatized by commercially available DMG and DMA charge labels in gentle condition. The specific neutral loss diagnostic ions were captured by mass spectrometry, which allowed enhanced ionization of various DAGs in comparison with conventional metal adducts. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the diagnostic ions of the derivatized DAGs have been developed for the detection and quantification of DAGs in serum. The derivative 1,2-DAGs and 1,3-DAGs


Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development.  Infections caused by antibiotic-resistant bacteria pose a serious challenge to healthcare systems worldwide. The increasing resistance of gram-positive and gram-negative pathogens causing infections in hospitals and in the general population and the worldwide spread of antibiotic-resistant 'superbugs’ represent a major global health problem,N-acetylglucosamine-enolpyruvyl transferase  is validated by fosfomycin, which is used to treat urinary tract infectionsPeptidoglycan is a macromolecule essential for bacterial survival and is found only in the bacterial cell wall. Therefore, enzymes involved in the peptidoglycan biosynthetic pathway represent potential targets for the discovery of new antimicrobial agentsN-acetylmuramoyl pentapeptideEscherichia coli, there are two isozymes of Ddl, DdlA and DdlB, which have similar catalytic efficiencies and substrate recognition properties, as well as similar sensitivity to inhibitors1 and thiosemicarbazone 2 . This approach is increasingly being used in the pharmaceutical industry and academia to reduce attrition and provide leads for previously inaccessible biological targets. FBDD identifies low molecular weight ligands (∼150 Da) that bind to biologically important macromolecules. The three-dimensional experimental-binding mode of these fragments is determined by X-ray crystallography or NMR spectroscopy and is used to facilitate their optimisation into potent lead compounds with drug-like properties by known methods such as fragment growing and fragment linking. Compared with high-throughput screening, the fragment-based approach requires a smaller number of compounds to be screened and provides more efficient and fruitful optimisation campaigns2+-dependent metalloenzyme, fragments capable of chelating metal ions could potentially bind to the active site via coordination interactions with Mg2+ ions. A subset of chelating fragments was designed using substructure filters for chelating groups (Supplementary Table S2),Supplementary Table S3) aiming to bind into the phosphate-binding site pockets normally occupied by ATPSupplementary Table S4). The final subset was designed by selecting the highest scoring hits obtained by docking a virtual library of available fragments to ATP- and D-Ala-binding sites. In total, the fragment library contained 943 compounds .The definition of fragments in this study was based on physicochemical properties, the most important criterion being a molecular weight not exceeding 300 Da. Half of the fragment library was designed for a general purpose and will be screened on other targets as well. It contained diverse fragments (purchased or available in-house) without reactive functional groups. The second half consisted of subsets specifically designed to target DdlB. Since the enzyme is a dinuclear Mgompounds  distribu50 determination (3 and 4).The fragment library was assayed in a DdlB inhibition assay, where fragments were tested for their ability to inhibit the D-Ala-adding activity of DdlB ligase. The orthophosphate formed during the enzymatic reaction was measured spectrophotometrically using malachite green reagent. The tested fragment concentrations varied from 1.5 to 5 mM. The criteria for classifying a fragment as a hit were: <50% residual DdlB activity and solubility (low background absorbance of the compound at 650 nm). The results are provided in a Supplementary excel file. We re-purchased six hit compounds that matched the criteria to determine the inhibition of DdlB from freshly prepared compound solutions . However4 and 5 were found to interfere with the assay by inhibiting MurA, DdlB, and binding phosphate , was used to detect compounds generating reactive oxygen species in the presence or absence of a reducing agent TCEP. The resazurin assay was used to detect redox active compounds that catalyse the conversion of resazurin to resorufin by the formation of free radicals in the presence of the reducing agent DTT. Compounds 4–6 and 8 were found to be active in at least one of the redox activity assays (Table S5). Because our goal was to discover noncovalently binding inhibitors of DdlB, the fragment hits were assayed for their potential reactivity with a nucleophilic thiol surrogate, reduced 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Fragment 4 was found to be reactive in this assay .To pursue only compounds with tractable mechanism of action, extensive profiling of fragment hits was performed. First, deselection was performed with a MurA inhibition assay using the same assay technology to find compounds that interfered with the malachite green assay system. Second, the fragment hits were tested in a phosphate-binding assay that omitted the enzyme and again used the same assay technology. Compounds hosphate . Redox a3 without red flags and with an IC50 value of 168 μM (50 = 108 μM). Compound 3 was part of a Docked fragments to DdlB subset of the fragment library. In particular, docking to the ATP-binding site was used in the selection of this compound as part of the fragment library design. Structurally, 3 is a pyrazolopyrimidinecarboxylic acid with a molecular weight of 253 Da.Based on the fragment hit profiling results, compounds with at least two red flags were excluded. For further evaluation, we selected fragment hit f 168 μM Table 2,3 were purchased  , even small changes may contribute to loss of inhibitory potencyIn an attempt to investigate the structure-activity relationship, 15 analogues of talogue) . Compoun3 to DdlB was confirmed by an orthogonal biophysical assay, surface plasmon resonance (SPR). The protein was covalently attached to the surface of the CM5 chip (∼7700 response units), and the ligand was titrated across the surface (KD = 3.2 mM) was estimated from the steady-state binding levels . The resulting best model showed that 3 is a competitive inhibitor with respect to ATP with a Ki value of 20.7 ± 4.5 µM. These data are in agreement with the docking results obtained during the design of fragment library, where 3 was identified as a potential ATP competitive inhibitor.Binding of  surface . The appg levels . Steady-3 was then additionally docked using extra-precision Glide (Glide XP)m-tolyl ring flip , with the largest fluctuations resulting from the flipping of the m-tolyl ring. The metal coordinating and ionic interactions with both of the active site Mg2+ ions through the carboxylate moiety and one pyrimidino nitrogen anchored 3 in the active site . Additionally, a cation-π interaction of the m-tolyl ring with Lys144 occurred quite frequently. Overall, in the absence of an experimental crystal structure, these results provide strong evidence for the proposed binding mode of compound 3 in the ATP-binding site.To confirm the relevance of the predicted docking pose, a 100 ns molecular dynamics (MD) simulation on the 3 were determined according to the European Committee for Antibacterial Susceptibility Testing recommendations and Clinical Laboratory Standards Institute protocolE. coli and Staphylococcus aureus and against two mutant E. coli strains. E. coli D22 bears a mutation in the LpxC gene that increases membrane permeability, whereas E. coli N43 is an AcrA knockout strain (knockout of the cell membrane pump). The 3 showed no antibacterial activity against both E. coli and S. aureus (Table S7). The most plausible reason is the low on target activity of the hit fragment, however, also the poor penetration into the bacterial cell or efflux pumps effect cannot be ruled out. Although the lack of antibacterial activity is a limitation, hit compound 3 represents a fragment-sized starting point for the development of new compounds with increased DdlB inhibitory potency as well as antibacterial activity.The antibacterial activities of 3 without flags and with an IC50 value of 168 μM was selected for further studies. We confirmed binding of 3 to DdlB with SPR analysis. Furthermore, steady-state kinetics showed 3 to be a competitive inhibitor with respect to ATP with a Ki value of 20.7 ± 4.5 µM. An analogue-by-catalogue campaign did not reveal more potent compounds, making fragment hit 3 a starting point for further synthetic optimisation to develop new antibacterial agents.Here, we describe the construction of a fragment library containing 943 fragments to discover novel inhibitors of a bacterial enzyme DdlB involved in intracellular steps of peptidoglycan biosynthesis. FBDD is an innovative and underutilised approach to target DdlB. The library contained diverse fragments suitable for screening on different targets, as well as fragments specifically designed to target DdlB. Library screening using a biochemical assay was followed by profiling of the fragment hits. Compounds that interfered with the assay, redox active compounds, and reactive compounds were flagged. The fragment hit in silico part of the fragment library preparation was performed using the KNIME analytics platformSupplementary Table S1) were applied using the SMARTS Query nodeThe The selected commercially available fragments were obtained at 1.0–1.2 mg on 96-well polypropylene plates. To each well, 100 μL of dimethyl sulfoxide (DMSO) was added, and the plates were covered with an aluminium seal, and sonicated to prepare 26–125 mM stock solutions, which were used for screening in a DdlB inhibition assay.Additional physicochemical filters were applied to the in-house available fragments, namely no more than 5 hydrogen bond acceptors, no more than 3 rotatable bonds, and a polar surface area greater than 0 and no greater than 80, resulting in a library of 887 in-house available fragments. Then, 400 diverse fragments were selected using the RDKit Diversity Picker node. The node uses a fast MaxMin algorithm to select diverse compounds based on the Tanimoto distance between the Daylight-like topological fingerprints. The diverse fragments were then manually inspected and a set of 195 fragments was selected for the physical fragment library. The fragments were prepared as 50 mM DMSO stock solutions and transferred to 96-well polypropylene plates.k-means clustering into 500 clusters was applied to the calculated distance matrix for Daylight-like topological fingerprints using the k-Medoids node. Next, we manually inspected 500 medoids and selected a set of 283 to be included in the physical fragment library.From the 91,379 commercially available fragments, 2000 diverse fragments were selected using the RDKit Diversity Picker node. Subsequently, Supplementary Table S1) without filters for phosphor and catechol was used in the previous step to prepare the subset of chelating fragments. Based on recent reviews,Supplementary Table S2) to find fragments capable of metal ions chelation. The RDKit Substructure Filter was used in the KNIME analytics platform. When filtering with a particular SMARTS pattern resulted in more than 100 fragment chelators, the RDKit Diversity Picker node was used to select 100 diverse fragment chelators. A total of 214 fragments were then manually selected.The Brenk filter (Supplementary Table S1) without filters for Michael acceptors was used in the previous step to prepare the subset of phosphate bioisosters. SMARTS patterns for phosphate bioisosteres were designed based on a reviewThe Brenk filter (Supplementary Table S4) to find fragments containing similar substructures to cycloserine. The RDKit Substructure Filter was used in the KNIME analytics platform. A total of 31 cycloserine analogues were manually selected.We applied several SMARTS patterns (www.eyesopen.com) was used to define grid boxes for each of the binding sites separately. The ATP binding site was defined with a box of 4214 Å3 (14.39 Å × 14.37 Å × 20.38 Å) and the outer contour of 739 Å3. The D-Ala binding site where cycloserine binds was defined with a box of 2155 Å3 (12.71 Å × 11.43 Å × 14.84 Å) and the outer contour of 308 Å3. The virtual fragment library containing 42,700 commercially available fragments was first processed using FixpKa  to obtain proper ionisation at pH 7.4. Subsequently, the stereoisomer and conformational model generator OMEGA 3.1.2.2  was used to enumerate stereocenters and to generate up to 200 conformers per compound. The prepared fragment library was then docked to each binding site individually using FRED 3.4.0.2 . Validation of the docking protocol was performed by redocking the co-crystallised ADP and D-cycloserine phosphate yielding RMSD < 2.0 Å. The 200 highest scoring virtual screening hits for the ATP binding site were clustered into 50 clusters, and 36 diverse fragments were manually selected. The 400 highest scoring virtual screening hits for the ATP binding site were clustered into 60 clusters, and 25 diverse fragments were manually selected.The final subset contained the highest scoring fragments from docking to DdlB. The DdlB protein was prepared from the X-ray structure in complex with ADP and D-cycloserine phosphate (PDB ID: 4C5A)E. coli DdlB enzyme  was expressed in E. coli2, 6.5 mM (NH4)2SO4, 10 mM KCl, 700 μM D-Ala, 100 μM ATP, purified DdlB , and the test compound dissolved in DMSO. The final DMSO concentration was 5%. After 20 min of incubation at 37 °C, the reaction was terminated by adding Biomol® reagent (100 μL) and after 5 min at room temperature the absorbance was measured at 650 nm using a microplate reader . A parallel experiment without the enzyme was performed to detect insoluble compounds under the assay conditions and subtracted from each measurement. All experiments were performed in duplicates. RAs were calculated with respect to blank experiments without tested compounds and with 5% DMSO. The final concentrations for fragment screening were 1.3–6.2 mM for purchased fragments and 2.5 mM for in-house available fragments. IC50 values were determined by measuring the residual activities in quadruplicate at seven different compound concentrations and calculated using in GraphPad Prism .A recombinant 3, Ki values were determined against DdlB from E. coli. Ki determinations were performed under similar conditions as described for DdlB inhibition assay, where different concentration of one substrate and fixed concentration of the other were used. The concentration of ATP  was varied at fixed concentration of D-Ala (700 µM). The concentrations of compound 3 were 0, 25, 50, 100, 250, 500, and 1000 µM. After 20 min of incubation, 100 µM of Biomol® green reagent was added, and absorbance was read at 650 nm after 5 min. Initial data were fitted to competitive, (v/V = [S]/{KM(1 + [I]/KI)+ [S]}), non-competitive (v/V = [S]/{(1 + [I]/KI)(KM+ [S])}), uncompetitive (v/V = [S]/{KM+ [S](1 + [I]/KI)}) inhibition models in which v is observed reaction rate, V is the maximum rate, KM is the Michaelis constant, using SigmaPlot 12.0 software. The best fit was taken as the one having the highest R2, the lowest standard error and the narrowest 95% confidence interval in each parameter (see Supplementary Table S6).For compound E. coli MurA enzyme (UDP-N-acetylglucosamine enolpyruvyl transferase) was expressed in E. coliN-acetylglucosamine, 100 μM phosphoenolpyruvate, purified MurA , and 5 mM of each test compound dissolved in DMSO. The final DMSO concentration was 5%. After 15 min of incubation at 37 °C, the reaction was terminated by adding Biomol® reagent (100 μL), and after 5 min at room temperature the absorbance was measured at 650 nm using a microplate reader . A parallel experiment without the enzyme was performed to detect insoluble compounds under the assay conditions and subtracted from each measurement. All experiments were performed in duplicates. RAs were calculated with respect to blank experiments without tested compounds and with 5% DMSO.A recombinant ® reagent was added and after 5 min, the absorbance was measured at 650 nm using a microplate reader . Background absorbance was determined in a parallel experiment without phosphate standard and subtracted from each measurement. To determine the blank value, the compound solution was replaced with pure DMSO and the background-subtracted measurements were divided by the blank value.Phosphate binding was determined by colorimetric measurement of phosphate with malachite green in the presence or absence of the compound. To 46.9 μL of buffer , 0.625 μL of 800 μM phosphate standard, and 2.5 μL of 100 mM compound DMSO stock was added. The reaction mixture was incubated for 5 min at room temperature. Then, 100 μL of BiomolThe HRP-PR assay was performed according to a previously optimised procedure2DCFDA assay was performed according to a previously optimised procedure2DCFDA was prepared as a 5 mM stock in DMSO and diluted 10-fold to 500 µM with 0.01 M NaOH. The probe was freshly prepared for each plate and stored in the dark at room temperature for 30 min to hydrolyse the ester. To 52.5 μL of buffer , 7.5 μL of 2 mM compound DMSO stock solution, 75 μL of buffer (redox-free) or 75 μL of 200 μM TCEP in buffer were added, and 15 μL of 500 µM H2DCFDA. The final concentrations were: 100 μM compound, 50 μM H2DCFDA, 100 μM TCEP, and 5% of DMSO. The microplate was covered with a lid and stored in the dark at room temperature for 30 min. Fluorescence intensity was then measured using 485 nm excitation and 535 nm emission filters . To determine the blank value, the compound solution was replaced with pure DMSO. The measured fluorescence for each compound was then divided by the blank value. 3-Methyltoxoflavin was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.The HThe resazurin assay was performed according to a previously optimised procedure2−in situ) in buffer  with 5% final DMSO concentration. To monitor TNB2− depletion, absorbance at 412 nm was measured every 5 min for 12 h . Compound background absorbance was subtracted from each measurement. 2-Chloro-N-(3-chlorophenyl)acetamide was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.The assay was performed as previously described3 compound with the DdlB protein we immobilised approximately 7700 RU of the purified DdlB protein on the carboxymethyl dextran-coated gold surface of the series S sensor chip CM5  to which DdlB was immobilised via the free amino groups by an amine coupling procedure3 was serially diluted in running buffer composed of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% P20. To assay the fragment–DdlB interaction, each fragment concentration tested (ranging from 78.125 to 5000 µM) for 40 s at a flow rate of 30 μL/min and dissociation was followed for 40 s. For the regeneration of the surface of the sensor chip, 2 mM NaOH was used. As the stock of the compound was prepared in 5% DMSO, we referenced the sensorgrams for the buffer response containing identical concentration of DMSO as the diluted sample of the compound. Furthermore, sensorgrams were referenced for the untreated surface flow-cell 1 response. The data were analysed with the Biacore T200 Evaluation Software and DK was determined by fitting the data to the steady state affinity model. The average DKs and standard deviations were determined from three titrations of 3.The SPR measurements were performed at the Infrastructural Centre for Analysis of Molecular Interactions at the Department of Biology, University of Ljubljana on a Biacore T200  at 25 °C. To assay the interaction of 3 were determined by broth microdilution in Cation-adjusted Mueller-Hinton broth (MH) with TES, TREK Diagnostic Systems Ltd. against S. aureus ATCC 29213, E. coli ATCC 25922, E. coli N43 and E. coli D22. All of the experiments were performed in duplicate in 96-well plate format according to CLSI guidelines and European Committee for Antimicrobial Susceptibility Testing recommendations. The bacterial suspension of a specific bacterial strain was diluted with MH broth to obtain a final inoculum of 5 × 105 CFU/mL in the assay. Compound 3 dissolved in DMSO and inoculum were mixed together and incubated for 20 h at 35 °C. After incubation, the minimal inhibitory concentration (MIC) values were determined by visual inspection as the lowest dilution of compounds showing no turbidity. The MICs were determined against S. aureus (ATCC 29213), E. coli (ATCC 25922), E. coli N43, and E. coli D22 bacterial strains. D-Cycloserine was used as a positive control.Minimum inhibitory concentrations (MICs) for 3 and outer box of 23.2 Å3. The structure of 3 was prepared with LigPrep, ionised with EpikThe DdlB protein was prepared from the X-ray structure in complex with ADP and D-Ala-D-Ala (PDB ID: 4C5C) using Protein Preparation Wizard3 was merged with the crystal structure, then the system was prepared with System Builder: TIP3P+ and Cl– ions were added to neutralise the system and produce the final 0.15 MFor molecular dynamics, the docking pose of d6 as a solvent. Chemical shifts are reported in parts per million (ppm), and the central peak of the residual solvent resonance at 2.50 ppm was used as the reference. The multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), ddd (doublet doublet of doublets), tt (triplet of triplets), dt (doublet of triplets), td (triplet of doublets), and qi (quartet of doublets), coupling constants (J) quoted in Hertz (Hz), number of equivalent nuclei (by integration). To further confirm the identity of compounds, high-resolution mass spectrometry measurements were performed on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer . Compound purity was determined by HPLC analysis on Thermo Scientific Dionex UltiMate 3000 modular system (Thermo Fisher Scientific Inc.) with Waters Acquity UPLC® HSS C18 SB column  thermostated at 40 °C, injection volume, 1 µL; flow rate, 0.3 mL/min; detector λ, 220 nm and 254 nm; mobile phase A: 0.1% TFA (v/v) in water; mobile phase B: MeCN. Method: 0–9 min, 5–95% B; 9–11 min, 95% B; 11–11.5 min, 95–5% B. Compounds are >95% pure by HPLC analysis, unless stated otherwise.Compound were purchased from several vendors  and used as received. The identity of compounds was confirmed with nuclear magnetic resonance, recorded on a Bruker Avance III 400 MHz spectrometer, using DMSO-1H NMR  δ 13.36 , 8.70 , 7.92 , 7.89 − 7.84 , 7.52 , 7.49 − 7.43 , 7.35 , 7.21 , 2.43 . 13C NMR  δ 163.95 (COOH), 151.16 (C5), 150.09 (C3), 148.02 (C1'), 146.70 (C7), 138.39 (C3'), 132.26 (C4'), 130.67 (C3a), 130.23 (C2'), 128.93 (C5'), 127.12 (C6'), 110.15 (C6), 99.38 (C2), 21.50 (CH3). HRMS (ESI+) m/z [M + H]+, calcd. for C14H12N3O2: 254.09240, found: 254.09159. Purity by HPLC: 95.6%. 2 D NMR spectra are shown in Supplementary Figures S6–S12.1H NMR  δ 8.63 , 8.01 − 7.90 , 7.68 − 7.58 , 7.57 − 7.47 , 6.67 , 4.74 , 4.57 , 4.26 , 3.96 , 3.68 , 2.97 − 2.80 , 1.30 . Purity by HPLC: 92.6%.1H NMR  δ 8.61 , 7.55 , 7.31 , 7.18 , 5.81 . Purity by HPLC: 98.5%.1H NMR  δ 12.46 , 8.86 , 8.63 , 8.15 − 8.06 , 7.70 − 7.57 , 7.46 . Purity by HPLC: 98.8%.1H NMR  δ 8.82 , 8.62 , 8.06 − 7.99 , 7.47 − 7.40 , 2.43 . Purity by HPLC: 98.0%.1H NMR  δ 12.40 , 8.81 , 8.63 , 7.92 − 7.82 , 7.42 , 7.38 , 2.34 , 2.33 . Purity by HPLC: 93.0%.1H NMR  δ 12.46 , 8.86 , 8.63 , 8.17 − 8.10 , 7.74 − 7.67 , 7.49 . Purity by HPLC: 98.6%.1H NMR  δ 12.50 , 8.98 , 8.91 , 8.67 , 8.53 − 8.45 , 7.93 , 7.62 . Purity by HPLC: 98.1%.1H NMR  δ 8.76 , 8.56 , 7.75 − 7.68 , 7.38 , 7.19 − 7.13 , 6.17 . Purity by HPLC: 98.2%.1H NMR  δ 13.51 , 8.68 , 8.58 , 8.14 , 7.87 , 7.41 , 7.20 . HRMS (ESI+) m/z [M + H]+, calcd. for C11H8N3O2S: 246.03317, found: 246.03262. Purity by HPLC: 99.3%.1H NMR  δ 12.43 , 9.16 , 8.81 , 8.70 , 8.08 , 7.82 , 7.76 . Purity by HPLC: 98.9%.Purity by HPLC: 95.7%.1H NMR  δ 13.45 , 8.69 , 8.58 , 8.14 , 7.88 , 7.41 , 7.23 . Purity by HPLC: 96.6%.1H NMR  δ 13.37 , 8.71 , 8.15 − 8.05 , 7.70 − 7.59 , 7.38 , 7.22 . Purity by HPLC: 96.9%.1H NMR  δ 8.85 , 8.67 , 7.91 − 7.82 , 7.45 , 7.38 , 4.32 , 2.34 , 2.33 , 1.33 . Purity by HPLC: 98.4%.1H NMR  δ 8.85 , 8.60 , 8.17 − 8.09 , 7.71 − 7.58 , 7.55 , 7.47 . Purity by HPLC: 99.0%.1H NMR  δ 8.92 − 8.84 , 7.93 − 7.85 , 7.59 − 7.44 , 2.42 . Purity by HPLC: 94.0%.1H NMR  δ 9.43 , 8.72 , 8.50 , 8.45 , 8.38 , 8.01 , 7.87 , 7.43 , 6.03 . Purity by HPLC: 98.5%.Click here for additional data file.


Pinus L. genus comprises around 250 species, being popular worldwide for their medicinal and aromatic properties. The present study aimed to evaluate the P. halepensis Mill. essential oil (PNO) in an Alzheimer’s disease (AD) environment as an anxiolytic and antidepressant agent. The AD-like symptoms were induced in Wistar male rats by intracerebroventricular administration of amyloid beta1-42 (Aβ1-42), and PNO (1% and 3%) was delivered to Aβ1-42 pre-treated rats via inhalation route for 21 consecutive days, 30 min before behavioral assessments. The obtained results indicate PNO’s potential to relieve anxious–depressive features and to restore redox imbalance in the rats exhibiting AD-like neuropsychiatric impairments. Moreover, PNO presented beneficial effects against neuroinflammation and neuroapoptosis in the Aβ1-42 rat AD model.The  Ranked as the fifth-leading cause of death worldwide , AlzheimAD is a pathologically heterogeneous and biologically multilayered disease characterized by gradual memory loss and cognitive and behavioral dysfunctions , currentThe occurrence of depression and anxiety in AD significantly affects the patient’s quality of life and function levels . Even thMedicinal plants represent a rich source of a valuable therapeutic biomolecule with the potential for ameliorating anxious–depressive symptoms , and havPinus halepensis, which appears as a potent medicinal plant with antioxidant [P. halepensis essential oil (PNO) [For ioxidant ,62, antiioxidant , and antioxidant  and hippioxidant , the antil (PNO) , this stThe PNO extraction procedure and the subsequent analysis of the obtained natural product have been previously described elsewhere .For the present study, a total of 70 three-month-old Wistar albino male rats, weighing 300 ± 15 g, were used. All the experimental animals have been accommodated in 1500 U Polysulfone cages (480 × 325 × 210 mm) with free access to water and standard certified rodent food, being housed in an artificial, ventilated, and thermally controlled room (22 °C) with a 12-h light/dark cycle (starting at 8:00 h in the morning). All of the in vivo experimental protocols have been established in agreement with the European Communities Council Directive (Directive 2010/63/EU) as well as the “Principles of Laboratory Animal Care” (NIH publication No. 85-23) regarding the protection of animals used for scientific and experimental purposes with approval of the Ethics Committee on Animal Research of the Alexandru Ioan Cuza University of Iasi, Faculty of Biology  under license no. 15309/22 July 2019.n = 10): (1) the untreated and untested group (naive); (2) the saline-treated group (sham-operated); (3) the group treated solely with Aβ1-42 (Aβ1-42); (4) the Aβ1-42 group also receiving diazepam (Diaz) (Aβ1-42 + Diaz); (5) the Aβ1-42 group also receiving imipramine (Imp) (Aβ1-42 + Imp); (6) the Aβ1-42 group also receiving 1% P. halepensis essential oil (Aβ1-42 + 1% PNO), and (7) the Aβ1-42 group also receiving 3% P. halepensis essential oil (Aβ1-42 + 3% PNO).Three days before the surgical interventions, the rats were randomly allocated to 7 experimental groups  in the third cerebral ventricle via a well-established, previously described protocol . The remThe i.p. route has been used to deliver imipramine   and diazTwo Plexiglass chambers (50 × 40 × 28 cm) were used for administration via inhalation by using an electronic vaporizer , placed at the bottom of the inhalation chambers. In one of the chambers, the rats corresponding to sham-operated, Aβ1-42, Aβ1-42 + Diaz, and Aβ1-42 + Imp groups were exposed to a 1% Tween 80 solution used for the dilution of the essential oils . The other chamber has been used to expose the rats corresponding to Aβ1-42 + 1% PNO and Aβ1-42 + 3% PNO groups to PNO in concentrations of 1% and 3%, respectively . The inhalation sessions were performed for 21 consecutive days, starting with day 5 poststereotaxic surgery and, on the test days, they were conducted 30 min before behavioral assessments . Each inAnxiety-like behavior has been assessed by using the elevated plus maze test (EPM) developed by Pellow et al. . A blackDepressive-like behavior has been assessed by using the forced swimming test (FST) developed by Porsolt , with soAfter behavioral assessments, all animals were euthanized by using a sodium pentobarbital overdose , humanely decapitated, and whole brains were collected. From five randomly selected rats per group, the brain regions containing amygdalas were carefully excised on ice, washed with ice-cold PBS  for blood removal, and stored at −20 °C until the protein extraction procedure was applied . From thg for 15 min and the supernatants containing proteins were collected. For the obtained amygdala protein extracts, the total protein content was determined via a bicinchoninic acid (BCA) protein assay kit  as previously described [The amygdala tissue samples were removed from −20 °C, slowly thawed on ice, weighed, and homogenized (1:10) in ice-cold 0.1 M potassium phosphate buffer (pH 7.4), containing 1.15% KCl. The amygdala homogenates were centrifuged at 960× escribed . The amy2O2 consumed/min/mg protein.For catalase  activity determination, a reaction mixture containing 150 μL phosphate buffer  and 100 μL amygdala protein extract was completed by the addition of 250 μL 0.16 M of a hydrogen peroxide substrate solution . The reaTo determine superoxide dismutase  activity, each 1.375 mL reaction mixture containing 1150 μL phosphate buffer (pH 7.8), 100 μL 0.1 M disodium EDTA (pH 7.8), 50 μL 1.5 mM NBT, 25 μL 0.12 mM riboflavin, and 50 μL amygdala protein extract was incubated at room temperature, monitoring the reduction of NBT to bluish-black formazan . To obta3 0.4 M, and 78 µL amygdala protein extract was incubated for 10 min at 37 °C. Each reaction mixture was completed by the addition of 50 µL of GSH 50 mM and 36 µL of H2O2 50 mM, which was followed by a secondary incubation step at 37 °C for 10 min. Finally, to each reaction mixture, 730 µL metaphosphoric acid 7% has been added and the reaction tubes were centrifuged at 14,000 rpm for 10 min. A total of 100 µL supernatant per reaction mixture has been mixed with 1270 µL disodium phosphate solution 0.3 M and 136 µL DTNB 0.04%, and the developed yellow color was read at 412 nm on a spectrophotometer. Control samples were prepared in the same manner, except that the amygdala protein extract was replaced with an equal volume of sodium phosphate buffer 0.25 M (pH 7.4). The GPX enzyme activity has been expressed as units/mg protein, where a GPX unit was defined as the amount of enzyme necessary to oxidize 1 µmol GSH/min.For the glutathione peroxidase  activity assessment,  each reaFor evaluation of reduced glutathione (GSH) content ,79, eachg. The obtained protein deposits were incubated with 500 μL 10 mM DNPH in 2 M hydrochloric acid for 60 min at 30 °C and stirred at 5-min intervals. Subsequently, 500 μL of 20% trichloroacetic acid was added to each protein deposit. After centrifugation at 960× g for 5 min, three washing steps with 1 mL ethanol:ethyl acetate (1:1) were performed. Next, the protein deposits were dried at room temperature and dissolved overnight in a 6-M guanidine hydrochloride solution in 20 mM monopotassium phosphate. The extinction of the resulting reaction mixtures was determined spectrophotometrically at 370 nm and expressed as nmol DNPH/mg protein.For determination of the carbonyl protein level , 1 mg ofg. For the control samples, the amygdala protein extracts were replaced with the same volume of phosphate buffer (pH 7.0). Finally, the reaction mixtures were spectrophotometrically determined at 532 nm, and the MDA level was expressed as nmol/mg protein.For the assessment of the malondialdehyde (MDA) level , 650 μL The relative quantification of histone-associated DNA fragments was performed by using a cell death detection ELISA kit version 8  as previously described . The wel® 1-Step RT-qPCR System  on a Rotor-Gene 6000 thermocycler , according to the manufacturer’s protocol. The absolute quantification for the transcripts of interest, ARC and IL1β, involved primers with the following sequences: IL-1β forward and reverse primer  , and ARC forward and reverse primer  . Data acquisition and analysis were performed by Rotor-Gene Q-Pure Detection Software v. 2.2.3. .The amygdala tissue samples preserved in RNA Save solution were removed from −80 °C, allowed to thaw at 4 °C, weighed, and homogenized in RNA lysis buffer. Total RNA was isolated from from the obtained amygdala homogenates by using the SV Total RNA Isolation System kit , respecting the protocol provided by the manufacturer and methods previously described . Reversep < 0.05.GraphPad Prism v9.1.0 software  has been used for statistical data analysis. One-way analysis of variance (ANOVA), followed by Tukey’s posthoc multiple comparison test was applied, and all data is expressed as the mean ± standard error of the mean (S.E.M.). The statistical significance was set at The management of neuropsychiatric impairments in AD proved to be quite problematic, especially because the evidence base for an appropriate pharmacological approach is limited and ambiguous. Anxious, depressive, and psychotic features, as well as apathy and agitation tend to be unresponsive to acetylcholinesterase inhibitors or memantine in AD cases; hence, antipsychotics, antidepressants, sedatives drugs, or anxiolytics are seen as a rational complementary therapy in AD and frequently prescribed . Howeverp = 0.0004) (p = 0.0004) or 3% PNO, respectively (p = 0.0012)  A. Howeve 0.0012) A. Regard 0.0012) B. Diazepp < 0.0001) (p < 0.0001) (p = 0.0003 for 1% PNO and p = 0.0022 for 3% PNO) (p = 0.0001 for 1% PNO and p = 0.0015 for 3% PNO)  A and inc 0.0001) B, both c 3% PNO) A as well 3% PNO) B. Imipraβ-caryophyllene, α-pinene, and myrcene [β-caryophyllene were described in a rat model induced by a chronic restraint stress procedure [β-caryophyllene’s potential to ameliorate depressive-like behaviors has been also reported in an experimentally induced diabetes mice model [β-caryophyllene has anxiolytic effects like those of diazepam [α-pinene too, in a mice schizophrenia model induced by dizocilpine administration [α-pinene is also recommended for management of sleep disorders associated with anxiety [The PNO potential to ameliorate anxious–depressive-like behaviors in an Aβ1-42-induced AD model is most likely mediated by the PNO major components, namely  myrcene , especiarocedure . β-caryoce model . Moreovediazepam . Myrcenediazepam . Anxietystration ; α-pinen anxiety .Arc is a plasticity-related gene, belonging to the immediate–early gene family [Arc gene modulation -signaling pathway has been demonstrated to be directly involved in AD progression, the levels of serum IL-1β serving as a stage indicator in the neurodegeneration process [β overexpression as compared to both sham-operated (p = 0.0088) and naïve rats (p = 0.0037) (p = 0.0109 for 1% PNO and p = 0.0204 for 3% PNO)   . However 3% PNO) . It is w 0.8887) .β-caryophyllene reduced neuroinflammation in a transgenic APP/PS1 mice AD model by decreasing the expression of the tumor necrosis factor-α (TNF-α) and IL-1β proinflammatory cytokines [α-pinene countered the inflammation Aβ1-42 associated by modulating TNF-α, IL-1β, and interleukin-6 (IL6) expressions in an AD-like model induced in male Wistar rats [3) and D-galactose administration [The anti-inflammatory properties of PNO may be attributed to its major components, for which neuroprotective properties were revealed in different murine AD-like models. It appears that ytokines . α-pinentar rats . Myrcenestration .p = 0.0084) (p = 0.0084) (p = 0.0103) (p = 0.0027) (p = 0.0066) (p = 0.0027) (p = 0.0238 for 1% PNO and p = 0.0047 for 3%PNO) (p = 0.0231 for 1% PNO and p = 0.0034 for 3% PNO) (p = 0.0053 for 1% PNO and p = 0.0037 for 3% PNO) (p = 0.0364) (p = 0.0051) (An ineffective antioxidant defense combined with reactive oxygen species (ROS) overproduction leads to oxidative stress, which is accounted as being responsible for cellular injury in aging and degenerative pathologies . The bra 0.0084) E and lip 0.0084) F rates,  0.0103) D and dec 0.0027) A, SOD (p 0.0066) B, and GP 0.0027) C. The inr 3%PNO) A and GPX 3% PNO) C activit 3% PNO) D in Aβ1- 0.0364) B. PNO in 0.0051) E. When r 0.0051) F.α-pinene demonstrated properties to combat AD-related oxidative stress through modulation of NF-E2-related factor 2/Kelch-like ECH-associated protein 1/antioxidant response element (Nrf2/Keap1/ARE) and interconnected pathways [β-caryophyllene seem to be linked to G-protein-coupled type 2 cannabinoid receptor (CB2R)-dependent Nrf2/heme oxygenase-1 (HO-1) antioxidant axis activation and inhibition of the HMG-CoA reductase activity [Targeting neuronal oxidative stress via therapeutics capable of not only passively scavenging radicals but also interfering with signal transduction pathways arose as a satisfying pharmacological approach in neurodegenerative disorders ,117. α-ppathways ,119. Myrpathways . Antioxiactivity ,122,123.p = 0.0026) and naïve rats (p = 0.0229) (p = 0.0129 for 1% PNO and p = 0.0265 for 3% PNO)  . However 3% PNO) .α-pinene and myrcene presented neuroprotective properties against apoptosis in different animal models of cerebral ischemia [β-caryophyllene showed anti-apoptotic properties in an in vitro model of Parkinson’s disease [The knowledge involving PNO’s main component effects regarding DNA fragmentation and cell death in AD is scarce. However, it appears that both ischemia ,128. On  disease .β gene expression and decreasing DNA fragmentation in the amygdalas of rats bearing AD-like neuropsychiatric deficits. Overall, based on the present findings, it can be concluded that PNO acts as potent multi-functional anti-AD agent. Finally, the biological mechanism by how PNO alleviate Aβ-induced neuronal dysfuctions can be summarized in The present experimental study has been designed to assess the anxiolytic and antidepressive properties of PNO in a rodent Aβ-induced AD model. Altogether, the obtained results provide evidence that PNO attenuates Aβ-related neuropsychiatric impairments, such as anxiety and depression, although these improvements appear to not be mediated via ARC modulation. Moreover, PNO restored the antioxidant defense system, upregulating antioxidant enzyme activity and reducing protein carbonylation at the amygdala level. In addition, PNO ameliorated both neuroinflammation and neuroapoptosis by downregulating IL1


The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β. These two isoforms are 85% identical and encoded by two different genes. Hsp90β is constitutively expressed and essential for early mouse development, while Hsp90α is stress-inducible and not necessary for survivability. These two isoforms are known to have largely overlapping functions and to interact with a large fraction of the proteome. To what extent there are isoform-specific functions at the protein level has only relatively recently begun to emerge. There are studies indicating that one isoform is more involved in the functionality of a specific tissue or cell type. Moreover, in many diseases, functionally altered cells appear to be more dependent on one particular isoform. This leaves space for designing therapeutic strategies in an isoform-specific way, which may overcome the unfavorable outcome of pan-Hsp90 inhibition encountered in previous clinical trials. For this to succeed, isoform-specific functions must be understood in more detail. In this review, we summarize the available information on isoform-specific functions of mammalian Hsp90 and connect it to possible clinical applications. Drosophila larvae [http://www.genenames.org/data/genegroup/#!/group/582; accessed on 5 August 2022) based on their observed molecular weights: Hsp70, Hsp90, Hsp40, the small HSPs, and chaperonins [Heat shock proteins (Hsps) are molecular chaperones which are known for their numerous roles in protein homeostasis (proteostasis), including protein folding and refolding, maturation, disassembly of aggregates, and degradation ,2,3. Thea larvae ,5. Latera larvae ,7. HSPs a larvae ,9. The hperonins ,11. All peronins . In thisArchaea and some bacterial species [Escherichia coli (E. coli) and humans, which is a strong indication that this protein has remained vital throughout evolution [Streptomyces coelicolor, contain another paralog, which shares only 30% identity [E. coli, although HtpG is not essential, it is relatively abundant under non-stress conditions and further induced during heat stress [E. coli htpG mutants have a growth defect at higher temperatures [Saccharomyces cerevisiae (S. cerevisiae), genome-wide studies suggested that up to 10% of all proteins are directly or indirectly dependent on Hsp90 for function [S.cerevisiae possesses two cytosolic Hsp90 isoforms encoded by separate genes, which arose from a genome duplication: the cognate Hsc82 and the stress-inducible Hsp82 [Homologous genes or proteins (homologs) are genes or proteins of different species with a common ancestor, whereas paralogs are genes with sequence homology that originate from the intragenomic duplication of an ancestral gene. For the Hsp90 family of proteins, there are homologs in all organisms except in  species ,14. Hsp9volution . During volution ,17,18. Tvolution ,17,18,19identity . In E. ct stress , and inderatures . During eratures ,24. The eratures ,24,25. Ifunction ,27. S.cle Hsp82 . Under nle Hsp82 . During le Hsp82 ,29. In mle Hsp82 ,32,33, Tle Hsp82 ,35, Grp9le Hsp82 , and chlle Hsp82 . Althougle Hsp82 ,17,18. IHSP90AA1 and HSP90AB1 genes, respectively [S. cerevisiae, Hsp90 isoforms readily form heterodimers both in vitro and in vivo [In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β . Human Hectively ,32,33. Hectively . These tectively . What maectively . Hsp90β ectively . Both isectively ,41. Inteectively . While tectively ,44, massectively  and sephectively . Whereas in vivo , this is in vivo . Hence,  in vivo ,39 , human brain has the highest expression of Hsp90α mRNA, and yet, this is not reflected at the protein level, as it is only moderate. For Hsp90β, there appears to be no such disparities . As in mice, human Hsp90β protein is moderately to highly expressed in all the major tissues. Hsp90α protein is more highly expressed in the respiratory system, and in female and male reproductive organs. These differences in tissue distribution patterns suggest that different tissues have distinct isoform-specific functional requirements. It is intriguing to speculate that different tissues might have different intrinsic levels of biophysical stress, due to differences in temperature, osmotic pressure, and oxygen availability, which both affect the differential expression of the two isoforms and impose distinct functional requirements.The expression levels of the two isoforms varies in a tissue-specific way. In mice, Hsp90β is highly abundant in heart, liver, spleen, lung, intestine, muscle, brain, testis, and kidney. In comparison, the levels of Hsp90α are lower in those tissues. However, in testis, retina, and brain, Hsp90α levels are comparatively higher than Hsp90β, whereas in heart and muscle, Hsp90α is almost absent ,49. IndeDuring evolution from prokaryotes to eukaryotes, overall proteome complexity dramatically increased without any accompanying gain of core molecular chaperones. To assist the core molecular chaperones and to diversify their functions, a large panel of co-chaperones appeared . Only rePost-translational modifications can have a large impact on the function and regulation of the two isoforms . Both isHSP90AB1 evolved as the constitutively expressed isoform, while HSP90AA1 evolved to be inducible in response to different types of stresses. The differential gene expression patterns of mammalian HSP90AA1 and HSP90AB1 were first characterized with transformed mouse cells [HSP90AB1 is constitutively expressed under normal conditions and has a 2.5-fold higher expression level than HSP90AA1. However, upon heat shock, HSP90AA1 expression increased 7-fold, while HSP90AB1 increased only 4.5-fold [HSP90AA1, there are several heat shock elements (HSEs) which enable the stress-mediated expression of Hsp90α [HSP90AB1 appears not to respond to heat shock [HSP90AB1 transcripts does not depend on HSF1 since hsf1 knockout oocytes do not show any reduction in Hsp90β mRNA [HSP90AB1 has a CAAT box, a specificity protein 1 (SP1) binding site, and a TATA box (−27 bp) [HSP90AA1 does not contain a CAAT box, but one has been identified far upstream at −1144 bp. The binding of Krüppel-Like-Factor 4 (KLF4) to the promoters of HSP90AB1 and HSP90AA1 leads to higher expression of both isoforms [HSP90AB1 by heat shock, STAT1 phosphorylation by Jak2 and PKC is necessary [HSP90AB1 expression is also regulated at the translational level by mTORC1 [se cells . Ullrich4.5-fold . This su4.5-fold . Other s4.5-fold ,68. Heat4.5-fold ,70. Upstf Hsp90α , and in f Hsp90α . Immediaat shock . Howeverat shock . In an a90β mRNA . The con(−27 bp) . The proisoforms . In addiisoforms . Interfeisoforms . For theecessary . Howevery mTORC1 . This sey mTORC1 ,78. Cleahttps://www.picard.ch/Hsp90Int, accessed on 5 August 2022 [https://www.picard.ch/downloads/Hsp90interactors.pdf, accessed on 5 August 2022. It is natural to assume that highly homologous versions of Hsp90s found in the same cellular compartment would have identical functions. However, these cytosolic Hsp90 isoforms have evolved to have some overlapping, synergistic, and distinct isoform-specific functions. In some contexts, they even have antagonistic functions (see below). Taipale and colleagues systematically characterized the chaperone/co-chaperone/client interaction network in human cells [S. cerevisiae by immunoprecipitation/mass spectrometry led to globally similar conclusions, except that it was noted that the vast majority of clients are shared by both isoforms [In humans, Hsp90α and Hsp90β together are predicted to interact with more than 2000 proteins . Not quiust 2022  and a doan cells . They prisoforms .In the next section of the discussion of isoform-specific functions and clinical relevance , we willHSP90AB1 gene has evolved to be expressed more or less constitutively, presumably to support essential cellular housekeeping activities. The embryonic lethality of the mouse knockout may be a reflection of that [During the course of evolution, the  of that . Hsp90β  of that . Thus, wHsp90β appears to have an exclusive role in muscle cell differentiation and regeneration in the mouse ,83. We sHsp90β is involved in controlling the formation of endodermal progenitor cells and development of the liver . For hepAnother important isoform-specific function of Hsp90β is regulating the responsiveness to vitamin D [Hsp90β is necessary for maintaining the neuromuscular junction (NMJ) . Rapsyn,As alluded to above, Hsp90β influences pathways regulating insulin resistance . It was Reduced levels of both Hsp90α and Hsp90β are associated with neuronal cell death in patients suffering from Alzheimer’s disease (AD) . HoweverListeria monocytogenes, suggesting that boosting Hsp90β levels and/or activity may protect against pathogenic infections. If so, EPO might also be beneficial against bacterial and viral infections.Hsp90β enhances the innate immune response . Hsp90β https://www.picard.ch/Hsp90Int, accessed on 5 August 2022), and yet the inhibition of Hsp90β, but not Hsp90α, led to the degradation of HIF1α in leukemia cells. This surprising result illustrated the potential of Hsp90 isoform-specific inhibition for the treatment of certain types of cancer.Although it is predominantly Hsp90α that is overexpressed in different types of cancer, in some cancers it is Hsp90β, which appears to be responsible for cancer cell survival ,101,102.In Ewing’s sarcoma, it was found that Hsp90β inhibition leads to decreased expression of the multidrug resistance-associated protein 1 associated with mitochondria . In laryMDR1 gene is responsible for exporting drugs from cells. Kim and colleagues showed that casein kinase 2 (CK2)-mediated phosphorylation of Hsp90β and the subsequent stabilization of its client PXR, a nuclear receptor, is a key mechanism in the regulation of MDR1 expression [Hsp90β plays a role in drug resistance in lung cancer. The P-glycoprotein (P-gp) encoded by the pression . Inhibitpression .Helicobacter pylori-induced gastric injury [H. pylori-induced translocation of Hsp90β to the membrane may ameliorate gastric injury. Nickel ions-mediated inflammation also occurs through Hsp90β in human B-cells. Nickel ions bind to the linker domain of Hsp90β and reduce its interaction with HIF1α. The released HIF1α then becomes more localized in the nucleus and enhances IL-8 expression [Possibly related to the dependence of liver cells on Hsp90β discussed above, Hepatitis B virus (HBV), which causes chronic infection in the liver, evades the immune defense by interaction with Hsp90β . Hsp90β c injury . Cha andpression .https://www.proteinatlas.org/search/HSP90AA1; accessed on 5 August 2022). For example, the brain has the highest levels of Hsp90α mRNA expression, which is several folds higher than any other organs. However, the highest levels of mRNA are not translated into proteins as brain expresses only moderate amounts of Hsp90α protein. A recent study says the human brain has a higher temperature than the usual body temperature ranging from 36.1 to 40.9 °C in a circadian way [The stress-inducible isoform Hsp90α helps cells adapt to stress . Most ofdian way . In addidian way . Mice widian way . Intrigudian way . The Hspdian way . The dowdian way ,119. It hsf1-/- oocytes [hsf1-/- females produce oocytes, they do not carry viable embryos. However, later we showed that no difference in embryo production was observed in female mice lacking Hsp90α compared to the wild-type [hsf1-/- oocytes failed to develop because of some Hsp90α-independent issue.Oogenesis may also be largely dependent on the Hsp90α isoform. Metchat and colleagues showed that extremely low levels of Hsp90α correlate with the developmental defects of  oocytes . While hild-type ,59. Henchttps://www.proteinatlas.org; accessed on 5 August 2022), which may be related to elevated local temperatures upon exposure to light. The local rise in temperature of human retina exposed to direct sunlight is about 2 °C [In the human retina, rod cells have high levels of Hsp90α mRNA expression . Peterson and colleagues showed that the potassium channel hERG, which is critical for cardiac repolarization, solely interacts with Hsp90α and not with Hsp90β [Muscle usually does not have much Hsp90α expression. However, among different type of muscle cells, cardiomyocytes have comparatively high Hsp90α mRNA expression (h Hsp90β . They foThe DNA damage response is assisted by Hsp90α. The DNA-dependent protein kinase (DNA-PK) is a component of the DNA repair machinery, and it is a client of both Hsp90α and Hsp90β ,125. HowHsp90α controls addictive behavior through the μ opioid receptor (MOR) . PreviouBesides functioning as an intracellular molecular chaperone, Hsp90α is also secreted from cells ,134,135.HSP90AA1 gene expression [HSP90AA1 expression in breast cancer cells through STAT5 [HSP90AA1 5′ flanking region, and not in that of HSP90AB1 [HSP90AA1 expression. In head and neck cancer cells, the transcription factor SOX11 binds to HSP90α [HSP90AB1 is also induced in certain cancers. However, the stress-inducible gene HSP90AA1 can be expressed several-fold higher than HSP90AB1. Thus, the balance of Hsp90α to Hsp90β is specifically shifted towards Hsp90α in cancer cells. It had been found that Hsp90α accounts for 2–3% of total cellular proteins in normal cells, but up to 7% in certain tumor cell lines [Specifically targeting the Hsp90α isoform could be an attractive therapeutic strategy for treating certain cancers ,140. Canpression . The grogh STAT5 . The nucgh STAT5 ,147. AmmHSP90AB1 . This mao HSP90α . In breao HSP90α ,151. As ll lines . Cancer ll lines ,154,155,ll lines . Althougll lines ,151,157.ll lines , and by ll lines . When Hsll lines ,162,163.ll lines ,165,166.ll lines . This suHsp90α plays a significant role in idiopathic pulmonary fibrosis (IPF) . BellayeWang and colleagues found that HSV-1 survive inside cells using Hsp90α of the host. Hsp90α stabilizes the virion protein 16 (VP16) and promotes VP16-mediated transactivation of HSV-α genes . When HsLoss-of-function mutations in the gene encoding the voltage-gated potassium channel KCNQ4 cause DFNA2, a subtype of autosomal dominant non-syndromic deafness characterized by progressive sensorineural hearing loss. The knockdowns of the two Hsp90 isoforms had opposite effects on the total KCNQ4 levels ,174. SpeHsp90α also has a connection with diabetes. High glucose was shown to induce the translocation of Hsp90α to the outside of aortic endothelial cells . In highIn contrast, the connection of Hsp90 with eNOS was found to be protective against the damages caused by ischemia-reperfusion by reducing the blood flow and glomerular filtration rate . When thElevated serum Hsp90α had been found in nonalcoholic steatohepatitis. Serum Hsp90α was increased in patients with metabolic-associated fatty liver disease (MAFLD). A positive correlation was found between age, glycosylated hemoglobin, serum Hsp90α, and grade of steatohepatitis . Xie andOverall, Hsp90α-dependent processes contribute to stress adaptation or other specialized functions, while Hsp90β is essential for maintaining standard cellular functions such as cell viability. We recently demonstrated that at cellular and tissue levels, albeit with some exceptions, it is the total Hsp90 levels that matter to sustain essential basic functions, without overt isoform-specific requirements . Pan-Hsp


Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly associated with poor survival in solid cancer patients and hence a low responsiveness to cancer immunotherapy. Therefore, 2′3′ Cyclic GMP-AMP (2′3′ cGAMP) was employed for its ability to shift macrophages from pro-tumoral M2-like macrophages (TAM) to anti-tumoral M1. However, cGAMP transfection within macrophages is limited by the molecule’s negative charge, poor stability and lack of targeting. To circumvent these barriers, we designed nanocarriers based on poly(amidoamine) dendrimers (PAMAM) grafted with D-glucuronic acid (Glu) for M2 mannose-mediated endocytosis. Two carriers were synthesized based on different dendrimers and complexed with cGAMP at different ratios. Orthogonal techniques were employed for synthesis , size , charge (DLS and NTA), complexation (HPLC-UV and AF4-UV) and biocompatibility and toxicity  evaluations in order to evaluate the best cGAMP carrier. The best formulation was selected for its low toxicity, biocompatibility, monodispersed distribution, affinity towards CD206 and ability to increase M1 (STAT1 and NOS2) and decrease M2 marker (MRC1) expression in macrophages. A hot tumor microenvironment (TME) is characterized by immune cell infiltration, consisting mainly of antigen-presenting cells (APCs) (mainly dendritic cell (DC) pro-inflammatory M1 subtype macrophages), T cells, and NK cells. These characteristics lead to responsiveness to immune therapy (particularly to immune checkpoint blockade) and overall better prognosis and patient survival. In contrast, a cold TME either lacks immune infiltration or is infiltrated by predominantly immunosuppressive tumor-associated macrophages (TAMs), which often exhibit an M2-like pro-tumor phenotype.Due to uncontrolled growth and aberrant microvasculature, there is increased oxygen consumption with decreased supply in the TME, which leads to the formation of hypoxic regions. When macrophages are recruited in hypoxic tumor regions, their polarization can be altered towards an M2-like pro-tumor phenotype via the activity of hypoxia-induced factors. Therefore, shifting the phenotype from pro-tumoral M2 to anti-tumoral M1 macrophages is an attractive strategy for many solid tumors that are highly infiltrated with TAMs, such as prostate cancer, pancreatic cancer, and glioblastoma, with the aim to increase the immune infiltration of anti-tumoral cells within the TME [A series of recent studies have indicated that STING pathway activation within macrophages results in a phenotype shift from the M2 to M1 polarization phenotype ,4. Moreo2′3′ cGAMP is a negatively charged dinucleotide due its phosphate groups; it therefore has limited transfection efficiency owning to the negative charge of cell membranes. Previous research has shown that intratumor (i.t.) administration is preferred compared to the intravenous (i.v.) route, due to the presence of enzymes in the blood stream, which would eventually degrade cGAMP . MoreovePAMAM dendrimers are suitable for condensing and thus carrying drugs and genes thanks to their great number of branches capped with primary amino groups, high drug loading capacity, and tunable size in the nanometer range ,10. BaseIl1b, Nos2 and Stat1—and for alternatively activated M2 macrophages, such as Mrc1, which codes CD206, Arg1 and Cdh1 [A series of recent studies have indicated that STING pathway activation within macrophages results in a phenotype shift from the M2 to M1 polarization status ,4. Thereand Cdh1 ,15. Moreand Cdh1 . CD206 iand Cdh1 ,18. Thisand Cdh1 . High exand Cdh1 . Sugars and Cdh1 . Since oand Cdh1 ,23. By dUnlike other studies, which encapsulate cGAMP to trigger immune responses within the TME, here we synthesized a carrier targeting M2 macrophages, which will additionally boost macrophage polarization . BesidesPAMAM with a 3rd generation (G3) and 4th generation (G4) ethylenediamine cores  were purchased from Sigma-Aldrich  D-Glucuronic acid (D Glu) from Alfa Aesar , and 2′3′-cGAMP was obtained from Invivogen . PAMAM-D glu is referred to in the following text as either PG3 or PG4 depending on whether it was synthesized using the above-mentioned G3 or G4 PAMAM.−1 mbar, −80 °C) before storage at 4 °C. Lyophilized PAMAM G3 or G4 was dissolved in distilled water at 0.1% (w/v) under magnetic stirring. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (5 eq. NH2), N-hydroxysuccinimide sodium salt (NHS) (2 eq. NH2) and D-Glucuronic  were added. pH was adjusted to 5.5 (827 pH Lab Meter from Metrohm) using NaOH/HCl. The reaction was allowed to proceed for 12 h under stirring at room temperature. The reaction mixture (G3 or G4 PAMAM-D-glucuronic acid) was dialyzed three times against 5% (w/v) NaCl  and three times against distilled water  and then lyophilized before storage at 4 °C. PAMAM G3 (20 wt% solution in methanol) or PAMAM G4 (10 wt% solution in methanol) was transferred into a round-bottom flask to evaporate the methanol using a rotary evaporator (337 mmHg at 40 °C for 20 min). In the same round-bottom flask, distilled water was added  and dry PAMAM was dissolved under magnetic stirring. The solution was then lyophilized   were assessed by  solvent ,25,26. D solvent ; Figure ® water.DS were confirmed via UV-vis spectroscopy using ninhydrin reagent. Ninhydrin assay was performed as previously described . The colw/v) in Milli-Q® water were determined on a HAAKE Mars Rheometer™  equipped with a cone-plate C35 2°/Ti measuring geometry on 420 µL samples. The complex viscosity (η*) was assessed at 25 °C and a constant oscillatory frequency of 1 Hz. Shear stress was set to 1 N/m2 in all experiments to stay in the linear viscoelastic region (LVE). Additional assays were performed with a frequency sweep from 0.1 Hz to 10 Hz. The assays were performed in triplicate on the day following the PG4 0.1 eq. and PG3 0.1 eq. solubilization. The rheological behaviors of PG4 0.1 eq. and PG3 0.1 eq. at different concentrations  on ≥10 mg samples.® water (n = 9), first for a PAMAM characterization alone at 0.1 mg/mL. Once NCs were prepared, they were measured at concentration 0.01 mg/mL of cGAMP in PG/cGAMP NC. The results are presented as mean ± SD of triplicate measurements of three different batches of PG3 and PG4.Z-average, polydispersity index (PDI) and zeta potential of PAMAM alone and PG/cGAMP NCs were measured by DLS and ELS : 1.331; 0.8872 cP, attenuator adjusted automatically). Size measurements were performed in ZEN0040 disposable cuvettes and zeta potential was measured using DTS1070 reusable cuvettes. Data were analyzed by Malvern Instruments Zetasizer Software version 7.13. Particle z-average, PDI and zeta potential were determined in Milli-Q® water (n = 3), which corresponds to cGAMP in PG/cGAMP NC, by nanoparticle tracking analysis . The cell used was ZetaView® NTA—ZNTA. The results are presented as mean ± SD of measurements of three different batches of PG3 and PG4.The size and zeta potential of PG/cGAMP NCs were analyzed at 0.01 mg/mL in Milli-QTo determine the recovery of cGAMP (%), radius of gyration (Rg), hydrodynamic radius (Rh), shape factor (Rg/Rh) and molecular weight (MW) of the PAMAM, PG and PG/cGAMP NC, we performed AF4-UV-MALS-DLS analysis . We used a new membrane (amphiphilic regenerated cellulose) that is suitable for cationic polymers with a 10 kDa molecular weight cut-off and a 350 µm spacer. The mobile phase was 0.05% Novachem . The particle size or gyration radius (Rg) and molecular weight were determined by multiangle light scattering (MALS), using Berry formalism, level 2, where linearity was obtained once 28° and 156° detectors were excluded. The hydrodynamic radius (Rh) or z-average was determined by AF4-DLS.® water. An amount of 50 µL of sample (5 µg) was manually injected. The method was optimized based on the Postnova® latex method for a duration of 25 min  and lyophilized . Recovery (%) and concentration (mg/mL) before and after filtration were investigated. PG3 and PG4 of different concentrations, from 0.05 mg/mL to 1 mg/mL, were placed on microscope slides , covered by cover slips  and analyzed in water by light microscopy . Microscopy was set at 10× magnification with phase contrast 10×.® water was placed on a copper grid and then vacuum-dehydrated on silica gel overnight. Samples were sputter-coated with 20 nm gold  and imaged by scanning electron microscopy .For imaging, 5 µL of 0.01 mg/mL PG/cGAMP NC in Milli-Qg over 20 min and the filtrate was analyzed [n = 3). Complexation efficiency (CE) was calculated as shown in Equation (2):PG/cGAMP NCs were separated from free cGAMP by centrifugation using VIVASPIN 3000 MWCO  at 4000× analyzed . cGAMP lA stock of cGAMP in LAL water (endotoxin-free water) was kept at 1 mg/mL at −20 °C. PG (PG3 and PG4) were freshly dissolved in water every time before complexation with cGAMP. Two N/P (PG/cGAMP NCs) ratios of 1/1 and 2/1 were used. The concentrations of cGAMP in PG/cGAMP NC suspensions were fixed at 0.01 mg/mL for physicochemical characterization, 0.25 mg/mL (5 µg per well) for in vitro, and 5 µg and 10 µg for in vivo experiments.As an example calculation, to obtain 0.010 mg/mL cGAMP concentration in PG/cGAMP NC, we added in equal parts a solution of 0.020 mg/mL of cGAMP and a solution of either 0.0125 mg/mL (ratio 1/1) or 0.0250 mg/mL (ratio 2/1) PAMAM. PG/cGAMP NCs were prepared by an ionic gelation method, as described previously for chitosan particles. PG was added dropwise to the cGAMP solution while vortexing for 30 s. Complexation was completed at room temperature (RT) over 30 min. At pH 6, both PG and cGAMP were fully ionized .PG/cGAMP NCs are labelled as either PG3 1/1, PG3 2/1 or PG4 1/1, PG4 2/1, depending on the polymer used as a carrier.A dye—Monolith His-Tag Labeling Kit RED-tris-NTA 2nd Generation (MO-L018) —was suspended at 5 µM in 1 × 20 mM HEPES (pH 7.4) supplemented with 0.1% pluronic F127. All further dilutions were also prepared in 1 × 20 mM HEPES (pH 7.4) supplemented with 0.1% pluronic F127.®), where the fluorescent signals of CD206 correspond to the typical detection limit of the Monolith NT.115 instrument . The mixture was incubated for 30 min at room temperature. After incubation, samples were centrifuged at 4 °C, at a speed of 15,000× g, and the supernatant was transferred to new PCR tubes. The final concentration of the fluorescently labelled CD206 protein was 4 nM. A total of 10 µL of ligand—glucuronic acid (and corresponding PG3 and PG4 concentrations)—was prepared in 16-tube serial 64—0.001953 M dilution. In each tube, 10 µL of previously prepared His-tagged CD206 protein was added to the ligand to a final volume of 20 μL using low-bind pipette tips. The samples were loaded into premium treated capillaries using capillary force. Three sets of six repetitions were performed on the NanoTemper® Monolith NT.115  in premium capillaries (MO-K025) at 25 °C using 40% MST power and 100% light-emitting diode (LED) with laser off/on times of 5 s and 30 s. The system was operated with the latest version of the MO control software (v1.6) with data analysis performed using the NanoTemper® analysis software. Results are presented as mean ± SD .Dye: 90 µL of 100 nM and 90 µL of 16 nM Recombinant Mouse Mrc1 (CD206) protein , supplemented with 1% Pen/Strep, 1% L-glutamine, 0.1% b-mercaptoethanol (50 mM) and 0.5% sodium pyruvate. Bone marrow cells were extracted from the femurs of C57BL/6J mice (Charles River) under license GE/33/20. The cells were treated with ACK lysis buffer for 3 min (Gibco) to remove red blood cells. Cells were then centrifuged, resuspended and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma), 1% antibiotic solution  (Gibco), MEM NEAA (Gibco), 10 mM HEPES (Gibco), 1 mM Sodium Pyruvate (Gibco), 50 µM 2-Mercaptoethanol (Gibco) and 10 ng/mL M-CSF (Immunotools) to induce differentiation into bone marrow-derived macrophages (BMDMs). The cells were cultured in two groups (M1 and M2) at 37 °C and 5% CO2. After 72 h post extraction, the original medium in all BMDM groups was aspirated to remove non-adherent cells and fresh medium was added to continue culturing. For the M1 group on day 5, IFN-γ (20 ng/mL) (Immunotools) was added for 1 h, followed by lipopolysaccharide (LPS) for 48 h (100 ng/mL). For the M2 group at day 5, IL-4 (20 ng/mL) (Immunotools) and IL-13 (20 ng/mL) (Immunotools) were added. On day 7 of culture, all the BMDMs were detached using TryLE Express buffer (Gibco) and the cells were subcultured on sterile 96-well cell-repellent plates (Cellstar).Bone marrow-derived dendritic cells (BMDC) were differentiated from C57BL/6JRj mice bone marrow cells by Dr. Julia Wagner. Cells were generated as previously described, with the only difference being that cells were split (1 to 2) at day 3. On day 6, BMDCs were seeded on a 96-well flat-bottom plate (Greiner) at a density of 1 × 10ulations ,31. BMDCA 96-well plate was coated with 1/625 diluted 50 µL/well anti-IFN β, RMMB-1  and incubated at 4 °C overnight;The next day, standard dilutions of mouse 1/840 dilution (from 1000 U/mL–7.8 U/mL) of IFN β  and samples were added ;Two hours later, the detection antibody, a 1/12,169 dilution of anti-IFN β , was added;Following this, the secondary antibody, anti-rabbit IgG, 1/1000 dilution HRP-linked , was added;Reaction was induced by the addition of TMB substrate ;2SO4;It was stopped by the addition of 25 µL HOptical absorbance was measured at 450 and 570 nm using a microplate reader (Biotek SynergyMx) and IFN beta levels are expressed as U/mL.BMDCs and M1 or M2 BMDMs were exposed to the different NCs (1/1 and 2/1 PG/cGAMP complexes made of PG3 and PG4) at 0.25 mg/mL (5 µg) cGAMP in formulation in half 0.9% NaCl and half respective cell culture media in a 96-well plate. After 24 h, supernatants were removed and assessed for Interferon β (IFN β) content and cells used for further flow cytometry measuring CD80/86. The protocol was as follows:n = 9; 3 wells per experiment 3×).Results are presented as mean ± SD of triplicate experiments  with the corresponding treatment . After 24 h, cells were collected and washed with 1 × PBS followed by blocking with CD16/CD32  for 15 min at 4 °C; they were then incubated with antibodies to obtain the proteins of interest , CD206-FITC, CD68-BV605, CD86 APC/Fire750, CD80-APC (Biolegend). For flow analysis, 1 × 10CD80-Pe (PE conjugated anti-mouse CD80 Antibody Biolegend);CD86-Bv510 ;Live/Dead Fixable Violet Dead Cell Stain (ThermoFisher)CD 206-FITC ;CD68-BV605 .Unstained cells were used to establish the flow cytometer settings. Fluorescence bead controls  were used for compensation. Flow cytometry data were acquired on a Gallios flow cytometer (Beckman Coulter), and the data were analyzed using Kaluza Software.w/v)), and finally resuspended in 100 µL FACS buffer. Results are presented as mean ± SD of triplicate experiments (n = 3).After incubation, cells were washed with FACS buffer  primers and PrimerScript reverse transcriptase enzyme —following the supplier’s instructions. Sybr green assays were designed using the program Primer Express v 2.0  with default parameters. Amplicon sequences were aligned against the mouse genome by BLAST to ensure that they were specific to the genes used. Arg1 , Mrc1 , Cdh1 , Il1b , Nos2  and Stat1 . Expression was calculated by normalization with Gapdh  and Eefa1a1 . Oligonucleotides were obtained from Invitrogen/Thermo Fisher. The efficiency of each design was tested with serial dilutions of cDNA. PCR reactions (10 µL volume) contained diluted cDNA, 2 × Power SYBR Green Master Mix (Applied Biosystems), 300 nM of forward and reverse primers. PCRs were performed on a SDS 7900 HT instrument (Applied Biosystems) with the following parameters: 50 °C for two minutes, 95 °C for ten minutes, and 45 cycles of 95 °C 15 s–60 °C. Each reaction was performed in three replicates on a 384-well plate. Raw Ct values obtained with SDS 2.2 (Applied Biosystems) were imported in Excel and the normalization factor and fold changes were calculated using the GeNorm method .No ethical approval was required for the chick embryo experiments, in accordance with the Swiss animal care guidelines . The experimental design took into consideration the 3R rules in order to improve animal welfare.Fertilized eggs from white hypex HN hens were obtained at the faculty animal facility .Upon receipt, eggs were carefully wiped with a paper using Milli-Q water then 70% EtOH. They were incubated on Embryonic Development Day 1 (EDD1) for 3 days with the apex down on the automatic rotation mode of the incubator  at 37.8 °C under 47% relative humidity. On EDD4, a 3–4 mm hole (in diameter) was drilled in the eggshell at the apex, closed with adhesive tape and incubated apex upwards in stationary mode for 7 days. On EDD11, the hole was enlarged with forceps to a diameter of ~3 cm to allow the treatment and imaging of the chorioallantoic membrane (CAM) vasculature.® and returned to incubate. Additional images were acquired on days D1, D2 and D3 post injection in order to assess the survival rates of embryos (n = 4) over 3 days.On EDD11, a volume of 20 µL of PG3 2/1, PG4 2/1 at 5 and 10 µg or control (WFI) was injected in the main blood vessel of the CAM by mean of a 33-gauge metal needle  mounted on a 100 µL glass syringe . Right after injection, CAM and embryos were imaged with an EOS 90D camera  equipped with an EF-S 18–135 mm f/3.5–5.6 IS USM lens  and mounted on a tripod. Acquisition parameters were set as follows: 1/100, F5.6, ISO 3200. Eggs were closed with Parafilmhttps://doi.org/10.5281/zenodo.5559119, accessed on 15 July 2022). The ROI was adjusted manually on each image.Images were batch cropped to 3000 px * 3000 px around the region of interest (ROI) using FIJI software and a cropping macro . Batch Cropping Images in ImageJ/Fiji. Zenodo. p < 0.05 was considered as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data were organized using Microsoft Excel  and statistical calculations were carried out using GraphPad Prism v. 7.0.2 . Values are given as mean ± SD.The results from multiple quantitative datasets where multiple variables were applied were analyzed using one- or two-way analysis of variance (ANOVA) followed by Tukey′s or Sidak’s multiple comparison test. PAMAM-Glu synthesis: Several chemical modifications of PAMAM dendrimers are well-described in the literature [terature ,33,34,35terature . D-glucuPAMAM-Glu synthesis: The water-based, green chemistry reaction for PG3 and PG4 is presented in 2 for ionic interaction with the STING ligand, cGAMP. The DS was calculated from two different complementary methods  (p < 0.05) to that of ninhydrin . Moreover, the gravimetry results followed the same trend for PG3 0.1 eq. and PG4 0.3 eq., but not PG4 0.1 eq. (7 nM) value was significantly lower compared to PG4 0.3 eq. (p < 0.01).n assay) b. Both Pn assay) a,b. The  0.1 eq. . However 0.1 eq.  and poss 0.1 eq. . Therefo 0.1 eq. . MoreoveDue to the better balance between blocked and free amino groups, offering sufficient binding sites for STING ligand–cGAMP complexation, a 0.1 equivalence was selected for further investigations. Moreover, PG3 0.1 eq. showed the highest CD206 affinity out of the three dendrimers examined, and consistent DS values with three measurement methods. Still, further investigations were carried out with both PG3 and PG4 0.1 eq. and all further preparations were prepared below their gelation concentrations.PAMAM-Glu complexation with cGAMP: Once PAMAM-Glu was synthesized, characterized and its reproducibility was assured, the next step was to complex it with the negatively charged cGAMP. Nanocomplexes (NCs) were prepared following the ionic gelation method at two different molar charge ratios (N/P), 1/1 and 2/1  and recovery (AF4/UV) of NCs were compared: CE% was around 20% and recovery was 10% for all NCs  were complexed with cGAMP . As a coIt is well-acknowledged that size, charge and shape are among the main aspects affecting the transfection performances of gene delivery systems, at least in vitro ,41. TherSize of NCs: NTA, as a number-based particle-by-particle method, was compared to an intensity-based (z-average) technique—DLS. DLS is sensitive towards big particles, while NTA gives the mean value of the whole population. If the sample is monodisperse, the intensity-based value would overlap with the number-based technique. In this work, dendrimers alone, both PG3 and PG4 controls, show significantly different NTA sizes compared to DLS (p < 0.01 for PG3 and p < 0.001 for PG4) and AF4-DLS (p < 0.0001), suggesting polydisperse and colloidally instable PG3 and PG4 samples  a;-p < 0.0001; PG4 2/1 < PG4 p < 0.01).PG NCs being significantly smaller by DLS compared to PG alone, especially in the case of the 2/1 ratio  almost equal to that of DLS—around 200 nm—while PG4 NCs were in range of 80–100 nm, which is smaller than their DLS and NTA sizes, especially in the case of 2/1 PG4 (p < 0.01), for which the polydispersity of the sample and possible aggregation might be the reason . Moreover, the pH of the PG3 2/1 suspension is (pH 6) most similar to the pH of the TME. For all NCs, zeta potential was significantly different between DLS and NTA (p < 0.01) (erformed b. When p < 0.01) .NCs′ shapes: By employing AF4-MALS-DLS, we obtained Rg (gyration radius—representing mass distribution) and Rh (hydrodynamic radius) values, and the so-called shape factor Rg/Rh. Based on shape factor, NCs can be classified as micro(nano)gels for values <0.7; homogeneous spheres for 0.775; branched molecules (1–1.5); random coils (1.5–2.1); and rod-like, elongated structures for values >2 [alues >2 . Based oConsidering size, charge, shape and complexation efficiency together, we may pre-select the most adequate(s) nanocomplexes. Besides the grafting of specific targeting moieties (D-Glu—CD206 Rc), we aimed for a particle size of around 200 nm to ensure efficient macrophage phagocytosis . We obseNCs do not cause toxicity: To investigate whether NCs can have direct cytotoxic effects on macrophages, we cultured M1 and M2 polarized BMDMs in 1% and 21% O2 with cGAMP, PG3 (1/1 or 2/1) or PG4 (1/1 or 2/1) NCs (2/1) NCs . After 2p > 0.99) toxic effects against M2 or M1 polarized BMDMs under normoxic (21% O2) conditions (2) in order to mimic TME milieu were not significantly (p > 0.99) affected by the NCs compared to the control group , we used Il1b, Nos2 and Stat1 gene expression signatures  genes and reduced the expression of the M2-associated gene gnatures . For altnd CCL18 . Indeed,rophages a as wellrophages . This mind Stat1 . Clearlyed BMDMs d,e. The ed BMDMs .Biocompatibility of NCs in chicken embryos: Considering that the 2/1 ratio was more effective compared to 1/1, in terms of its ability to control macrophage phenotype from M2 to M1, especially in the case of PG3, our final experiments used the 2/1 ratio. However, the last biocompatibility examination was performed before deciding which dendrimer will be the carrier of choice  at 1 min and 1, 2 and 3 days after an injection of 20 µL of WFI, 5 µg or 10 µg of PG3 2/1 or PG4 2/1 a. We obsConsidering that PG3 2/1 did not express any unusual phenomena at the site of injection, PG3 2/1 blood concentrations on injection day (EDD11) were estimated to be 3.731 and 7.463 µg/mL for 5 and 10 µg doses, respectively, and considered biocompatible and non-toxic .The results obtained in this work show that PG is a promising cGAMP transfection carrier. The characterization process provided insightful information about the polymer’s behavior in solution. The aggregation of PG3 and PG4 at a high concentration was increased, as confirmed by the increase in viscosity found using rheological measurements and from light microscopy observations. It is therefore important to prepare PG solutions below the gelation concentration. Complexation between PG and cGAMP was indirectly proven by size and zeta potential measurements, where particles were smaller and less charged compared to empty NCs. Moreover, AF4-UV and UPLC-UV showed 10 and 20% cGAMP recovery and complexation efficiency in NCs, respectively. Based on the in-depth orthogonal NC characterization by DLS, NTA, SEM, and AF4-MALS-DLS, the PG3 NCs seemed to be more stable and monodispersed, showing less aggregation and larger particles (200 nm) compared to PG4 NCs. Additionally, the PG3 carrier showed a higher affinity towards CD206, and the PG3 2/1 NCs demonstrated a clear effect increasing the expression of NOS1 and STAT1 genes characteristic for M1 macrophages, downregulating the expression of ARG1 and MRC1, characteristic for M2 genes and increasing IFN-β cytokine in SN in both M1 and M2 polarized BMDMs. Finally, PG3 2/1 was the condition that caused the fewest side effects on chicken embryos at both examined doses (5 and 10 µg). This work shows that decorating PAMAM with CD206 ligand such as glucuronic acid has potential as a viable M2 targeting transfection carrier for local cGAMP delivery to the tumor microenvironment of cold tumors.


However, antibody levels following 2nd, 3rd, and 4th dose SARS-CoV-2 vaccination over time in a cohort of IBD patients.We assessed the durability of serological responses to 2n=559) were evaluated for serological response to the spike protein of SARS-CoV-2 using the Abbott IgG II Quant assay with a seroconversion threshold of ≥ 50 AU/mL. The geometric mean titer (GMT) with 95% confidence intervals (CI) were calculated and stratified by weeks  after each vaccine dose. We compared stratified GMTs with Mann–Whitney U tests using a significance level of 0.05.Adults with IBD who received at least one dose of a SARS-CoV-2 vaccine (n=559) comprised the following patient characteristics: 82.8% were 18–65 years-old (n = 463), 53.1% were female (n =297), and 71.6% had Crohn’s disease (n =400). IBD medications were classified in the following mutually exclusive groups: No immunosuppressives 10.5% (n = 59), anti-TNF monotherapy 35.8% (n = 200), immunomodulatory monotherapy 2.1% (n =12 ), vedolizumab 11.8% (n =66 ), ustekinumab 20.4% (n =114 ), tofacitinib 1.2% (n =7 ), combination therapy 15.9% (n = 89), and prednisone 2.1% (n =12). For vaccine type, 85.6% and 82.3% had Pfizer for 3rd and 4th dose, respectively, while the remainder had Moderna. Seroconversion rates 1–8 weeks after 3rd and 4th dose were both 99.9%.Our cohort , 3rd dose , and 4th dose . Subsequently, antibody levels decay from 1–8 weeks to 8–16 weeks (p<0.001) for 2nd dose (mean difference: –2224 AU/mL), 3rd dose (mean difference: –7526 AU/mL), and 4th dose (mean difference: –9715 AU/mL). Compared to 16–24 weeks after 2nd dose, antibody levels 24+ weeks after were similar . For third dose, antibody levels 8–16 weeks and 16–24 weeks after vaccination were similar  along with 16–24 weeks compared to 24+ weeks after vaccination .Figure 1 compares GMTs with 95% CI by weeks after each vaccine dose. GMTs are highest 1–8 weeks after 2Within 1–8 weeks after each dose of vaccine, serological responses spikes with each subsequent dose yielding a higher GMT. While antibody levels decay 8–16 weeks after each dose, similar GMT levels beyond 16 weeks may indicate durability of antibody levels over a longer duration of time.None Declared