Peptide 1 has proven effective in experimental models of PTB and OIR. Seeking understanding of the structural requirements for the activity and biased signaling of 1, a panel of twelve derivatives was synthesized employing the various stereochemical isomers of α-amino-γ-lactam (Agl) and α-amino-β-hydroxy-γ-lactam (Hgl) residues to constrain the D-Thr-D-Val dipeptide residue. Using circular dichroism spectroscopy, the peptide conformation in solution was observed to be contingent on Agl, Hgl, and Val stereochemistry. Moreover, the lactam mimic structure and configuration influenced biased IL-1 signaling in an in vitro panel of cellular assays as well as in vivo activity in murine models of PTB and OIR. Remarkably, all Agl and Hgl analogs of peptide 1 did not inhibit NF-κB signaling but blocked other pathways, such as JNK and ROCK2 phosphorylation contingent on structure and configuration. Efficacy in preventing preterm labor correlated with a capacity to block IL-1β-induced IL-1β synthesis. Furthermore, the importance of inhibition of JNK and ROCK2 phosphorylation for enhanced activity was highlighted for prevention of vaso-obliteration in the OIR model. Taken together, lactam mimic structure and stereochemistry strongly influenced conformation and biased signaling. Selective modulation of IL-1 signaling was proven to be particularly beneficial for curbing inflammation in models of preterm labor and retinopathy of prematurity (ROP). A class of biased ligands has been created with potential to serve as selective probes for studying IL-1 signaling in disease. Moreover, the small peptide mimic prototypes are promising leads for developing immunomodulatory therapies with easier administration and maintenance of beneficial effects of NF-κB signaling.Interleukin-1β (IL-1β) binds to the IL-1 receptor (IL-1R) and is a key cytokine mediator of inflammasome activation. IL-1β signaling leads to parturition in preterm birth (PTB) and contributes to the retinal vaso-obliteration characteristic of oxygen-induced retinopathy (OIR) of premature infants. Therapeutics targeting IL-1β and IL-1R are approved to treat rheumatoid arthritis; however, all are large proteins with clinical limitations including immunosuppression, due in part to inhibition of NF-κB signaling, which is required for immuno-vigilance and cytoprotection. The all-D-amino acid peptide  In contrast to currently approved IL-1 inhibitors, peptide 1 exhibited pharmacological selectivity by inhibiting some IL-1-triggered pathways such as p38 mitogen-activated protein kinases (p38 MAPK) and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), while preserving NF-κB , and for preventing the premature initiation of uterine activation proteins and subsequent onset of labor in mice -Agl residue, respectively at either the D-Tyr, D-Val, D-Glu, or D-Leu positions demonstrated similar efficacy, and [(S)-Bgl4]-1 increased activity compared to peptide 1 in inhibiting IL-1β-induced DNA primase-1-dependent thymocyte TF-1 proliferation -Agl3]-1 exhibited significantly diminished activity, likely due to loss of side chain  and oxygen-induced retinopathy (OIR). These investigations have illustrated the influences of the orientation of the hydroxyl group and backbone for activity and biased signaling, particularly with respect to inhibition of NF-κB.Employing all configurations of Agl, Hgl, and Val residues to study the 4 or Na2SO4, filtered, and rotary-evaporated under reduced pressure. The syntheses under microwave conditions were performed on a 0–400 W Biotage® Robot Eight and Robot Sixty microwave synthesizer. Column chromatography was performed on 230–400 mesh silica gel, and thin-layer chromatography was performed on alumina plates coated with silica gel (Merck 60 F254 plates). Visualization of the developed chromatogram was performed by UV absorbance or staining with iodine or potassium permanganate solutions. Melting points were obtained on a Buchi melting point B-540 apparatus and are uncorrected. Specific rotations, [α]D values, were calculated from optical rotations measured at 20 and 25°C in CHCl3 or MeOH at the specified concentrations (c in g/100 mL) using a 0.5 dm cell length (l) on an Anton Paar Polarimeter, MCP 200 at 589 nm, using the following general formula: [α]c). Accurate mass measurements were performed on an LC-MSD instrument in electrospray ionization (ESI-TOF) mode at the Université de Montréal Mass Spectrometry facility. Sodium adducts [M + Na]+ were used for empirical formula confirmation. Nuclear magnetic resonance spectra  were recorded on Bruker 300, 400, 500, and 700 MHz spectrometers. 1H NMR spectra were referenced to CDCl3 (7.26 ppm), CD3OD (3.31 ppm), C6D6 (7.16 ppm) or DMSO-d6 (2.50 ppm), and 13C NMR spectra were measured in CDCl3 (77.16 ppm), CD3OD (49.0 ppm), C6D6 (128.39 ppm) or DMSO-d6 (39.52 ppm) as specified below. Coupling constant J values were measured in Hertz (Hz) and chemical shift values in parts per million (ppm). Infrared spectra were recorded in the neat on a Perkin Elmer Spectrometer FT-IR instrument, and are reported in reciprocal centimeters (cm−1). Analytical LCMS and HPLC analyses were performed on a 5 μM, 50 mm × 4.6 mm C18 Phenomenex Gemini column™ with a flow rate of 0.5 mL/min using appropriate gradients from pure water containing 0.1% formic acid (FA), to mixtures with either CH3CN containing 0.1% FA, or MeOH containing 0.1% FA. Peptides were purified on a preparative column (C18 Gemini column™) using appropriate gradients from pure water containing 0.1% FA to mixtures with MeOH containing 0.1% FA at a flow rate of 10 mL/min.Unless otherwise specified, all non-aqueous reactions were performed under an inert argon atmosphere. All glassware was dried with a flame under flushing argon gas or stored in the oven, and let cool under an inert atmosphere prior to use. Anhydrous solvents  were obtained by passage through solvent filtration systems  and solvents were transferred by syringe. Reaction mixture solutions (after aqueous workup) were dried over anhydrous MgSO3, DIAD, p-nitrobenzoic acid, NaN3, piperidine, DIEA, TFA, TES, TEA, CH3I, NaH, Fmoc-OSu, Boc2O, NaIO4, m-CPBA, TFE, and HFIP. Polystyrene Rink amide resin (0.5 mmol/g) was purchased from Advanced Chemtech™, and the manufacturer's reported resin loading was used in the calculation of yields of final product. All commercially available amino acids  and coupling reagents  were purchased from GL Biochem™ and used as received. Solvents were obtained from VWR international.Unless specified otherwise, commercially available reagents were purchased from Aldrich, A & C American Chemicals Ltd., Fluka and Advanced Chemtech™ and used without further purification, including PPhH-d-Arg-dR)-Agl--Tyr--Agl[(33R)-5]]-1,  and ; HRMS (ESI+) calcd m/z for C38H62N11O10 [M+H]+, 832.4676 found 832.4682.A 10 mL plastic filtration tube equipped with a polyethylene filter was charged with polystyrene Rink amide resin, 75–100 mesh, 1%, DVB with a 0.5 mmol/g loading, (200 mg) and DCM (about 7 mL). The tube was sealed, shaken for 30 min to induce swelling and the liquid phase was removed. The Fmoc group was cleaved from the resin-bound peptide by treatment with a freshly-prepared 20% piperidine in DMF solution (about 7 mL), shaking for 30 min, and removal of the liquid phase. The resin was repeatedly (3x per solvent) washed (10 mL per wash) over a total of 6 min with DMF and DCM, and the liquid phase was removed. Generation of the free amine resin was confirmed by a positive Kaiser test. Peptide elongation was conducted by treating the DMF-swollen free amine resin with a freshly prepared acylation solution containing Fmoc-D-Ala-OH (3 eq), HBTU (3 eq), and DIEA (6 eq) in DMF (4–7 mL). After agitating for 3–5 h at room temperature, Fmoc cleavage and peptide elongation were reiterated using Fmoc-D-Leu-OH, Fmoc-D-Glu-2-[3-N-(Fmoc)amino-2-oxopyrrolidin-1-yl]-3-methylbutanoic acid [-7]-Acid -14  was dissolved in 1:1 acetone (1 mL) and H2O (1 mL), treated with Na2CO3  followed by Fmoc-OSu , stirred at rt for 18 h, and concentrated to a residue that was dissolved in H2O and acidified to pH 3–4 with citric acid (0.5 N solution). The aqueous solution was extracted multiple times with EtOAc until the aqueous layer no longer exhibited the carbamate by TLC. The organic layer was extracted with saturated NaHCO3 (2 × 3 mL). The aqueous extractions were combined, acidified to pH 3–4 with 0.5 N citric acid solution, and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated to afford -carbamate -7 : Rf = 0.26 (10% MeOH:DCM); [α]c 0.41, MeOH); 1H NMR  δ 7.80 , 7.68 , 7.40 , 7.31 , 5.60 , 4.53–4.18 , 3.74 , 3.49–3.35 , 2.56–2.37 , 2.32–2.16 , 2.10–1.85 , 1.44–1.21 , 1.03 , 0.93 ; 13C NMR  δ 173.9, 171.6, 157.2, 143.9, 141.2, 127.4, 126.8, 124.8, 119.5, 66.7, 60.8, 52.2, 41.2, 28.1, 26.2, 18.4, 18.1; HRMS (ESI+) calcd m/z for C24H27N2O5 [M+H]+, 423.1914 found 423.1917.(3R)-N-(Boc)-Methioninyl-(R)-valine tert-butyl ester [-10]N-Boc-(R)-Methionine  and (R)-valine tert-butyl ester hydrochloride  were dissolved in dry DMF (6 mL). On treatment of the mixture with triethyl amine  a precipitate was formed, and HOBt  and DCC  were added to the mixture. The mixture was stirred at rt for 24 h, filtered and the filter cake was washed with DCM. The filtrate and washings were combined concentrated under vacuum. The residue was dissolved in EtOAc (6 mL), washed with 0.5 M citric acid (3 × 4 mL), 2 N aqueous NaHCO3 (3 × 4 mL) and brine (4 mL), dried over Na2SO4, filtered and concentrated under vacuum to give -dipeptide -10 as a white foam : Rf = 0.28 (20% EtOAc in hexane); [α] c 1.95, CHCl3); 1H NMR  δ 6.61 , 5.20 , 4.40 , 4.35-4.22 , 2.59 , 2.23-1.86 , 1.46 , 1.44 , 0.91 ; 13C NMR  δ 171.4, 170.7, 155.6, 82.1, 80.2, 57.7, 53.4, 31.6, 31.4, 30.3, 28.4, 28.2, 19.1, 17.6, 15.3; HRMS (ESI+) calcd m/z for C19H36N2O5S [M+H]+, 405.2418 found 405.2433.N-Boc-(R)-Methioninyl-(R)-valine tert-butyl ester methylsulfonium iodide [-11]R,R)-Dipeptide -10  was dissolved in CH3I  and stirred at rt for 24 h, and concentrated under vacuum. The volatile contaminants were removed by repeated (3x) co-evaporation with DCM to give -methylsulfonium iodide -11 as yellow gummy foam : Rf = 0.2 (5% MeOH in DCM); [α] c 1, MeOH); 1H NMR  δ 7.53 , 5.98 , 4.60 , 4.24 , 3.96-3.81 , 3.73–3.58 , 3.32 , 3.21 , 2.64–2.47 , 2.42–2.14 , 1.45 , 1.42 , 0.99 ; HRMS (ESI+) calcd m/z for C20H39N2O5S [M]+, 419.2574 found 419.2567.-2-[3-(Fmoc)amino-4-hydroxy-2-oxopyrrolidin-1-yl]-3-methylbutanoate [-12] was prepared as previously described -tert-Butyl 2-[3-(Boc)amino-2-oxopyrrolidin-1-yl]-3-methylbutanoate [-13]-Methylsulfonium iodide -11  was dissolved in a 1:1 mixture of DMF (3.5 mL) and DCM (3.5 mL) under argon, cooled to 0°C, treated with NaH , and stirred at 0°C for 2.5 h. The reaction was diluted with methyl acetate (2.5 mL) and H2O (0.5 mL) and allowed to warm to rt with stirring overnight. The reaction mixture was concentrated under vacuum, quenched with 1 M NaH2PO4, and extracted three times with EtOAc. The combined EtOAc layers were dried over MgSO4, filtered and concentrated to a residue that was purified by column chromatography on silica gel using 20% of EtOAc in hexane as eluent. Evaporation of the collected fractions provided -lactam -13 : Rf = 0.31 (30% EtOAc in hexane); [α] c 1.6, CHCl3); 1H NMR  δ 5.09 , 4.39 , 4.31–4.16 , 3.72 , 3.27 , 2.72–2.58 , 2.26–2.11 , 1.90–1.71 , 1.45 , 1.44 , 0.98 , 0.89 ; 13C NMR  δ 173.0, 169.7, 156.1, 82.1, 80.0, 61.0, 52.4, 41.7, 29.4, 28.8, 28.5, 28.2, 19.4, 19.3; HRMS (ESI+) calcd m/z for C18H33N2O5 [M+H]+, 357.2384 found 357.2399.-2-[3-Amino-2-oxopyrrolidin-1-yl]-3-methylbutanoate trifluoroacetate [-14]-lactam -13  in TFA (1 mL) and DCM (1 mL) was stirred at rt. After TLC analysis revealed complete consumption of the carbamate, the volatiles were evaporated on a rotary evaporator, and the residue was precipitated from ice-cooled diethyl ether and collected using a centrifuge to yield -trifluoroacetate -14  as white precipitate: Rf = 0.1 (1:9:90 Et3N:MeOH:DCM); [α]c 0.5, MeOH); 1H NMR  δ 4.40 , 4.17 , 3.92–3.81 , 3.60–3.45 , 2.66–2.54 , 2.35–2.19 , 2.14–1.97 , 1.05 , 0.98 ; 13C NMR  δ 171.3, 170.0, 60.8, 50.3, 41.6, 28.2, 24.5, 18.3, 18.2; HRMS (ESI+) calcd m/z for C9H17N2O3 [M+H]+, 201.1234 found 201.1226.A solution of (3N-Fmoc-(R)-Methionine methyl ester [(R)-15] and N-Fmoc-(R)-Methionine sulfoxide methyl ester [(R)-16] were prepared by a modified version (vide infra) of the previously described method -Vinylglycine methyl ester [(R)-17] and - and -Methyl 2-(oxiranyl)-N-(Fmoc)glycinate [-18 and -18] were prepared as previously described -Hgl-Val-tBu [-19] and -tert-Butyl 2-[3-(Fmoc)Amino-4-p-nitrobenzoyloxy-2-oxopyrrolidin-1-yl]-3-methylbutanoate [-20] were prepared as previously described -Agl3t-Bu)-D-Leu-D-Ala-Rink amide resin (22)]-D-Val-D-Glu-7  and 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate  in dimethylformamide  was stirred for 1 min, treated with N,N-diisopropylethylamine , stirred for 5 min and added to H-D-Glu(t-Bu)-D-Leu-D-Ala-Rink amide resin 21 , which was placed earlier into a 3-mL plastic filtration tube equipped with a polyethylene filter and swollen in DMF (2 mL). The resin mixture was shaken at rt for 5 h, and the liquid phase was removed by filtration. The resin was repeatedly (3x per solvent) washed (15 s per wash) with DMF and DCM, and dried under vacuum to afford resin 22. To assess resin-bound peptide purity, a resin aliquot (5 mg) was placed into a 1-mL plastic filtration tube equipped with a polyethylene filter and treated with 20% piperidine in DMF. After 0.5 h, the liquid phase was removed by filtration and the resin was treated with 0.5 mL of a 95:2.5:2.5 cocktail of TFA:H2O:triethylsilane (TES) at rt for 1 h, and filtered. The filtrate was collected in a 1.5 mL tube and concentrated by purging with an air stream. The residue was treated with Et2O (1 mL), and centrifuged for 2 min. After decantation, the precipitate was dissolved in H2O (1 mL) and analyzed by LCMS  and .A solution of acid  using a 1.0 cm path-length quartz cell containing 20 μM of peptide dissolved in Milli-Q water. The experimental settings were as follows: 1 nm, bandwidth; 0.5 nm, step size; 3 s, sampling time.Timed-pregnant CD-1 mice were obtained from Charles River  at gestational day 12 and were acclimatized for 4 days prior to experiments. Animals were used according to a protocol approved by the Animal Care Committee of Hôpital Sainte-Justine in accordance with the principles of the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The animals were maintained on standard laboratory chow under a 12 h:12 h light/dark cycle and allowed free access to chow and water.ad libitum access to food and water and kept in a 12 h:12 h light/dark cycle. To control for the effects of litter size on retinal development, the sizes of litters were reduced to 12 by sacrificing excess pups by decapitation while under 2% isoflurane anesthesia. All procedures and protocols involving the use of these rats were approved by the Animal Care Committee of the research center of Hôpital Maisonneuve-Rosemont and are in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research approved by the Association for Research in Vision and Ophthalmology (ARVO), and guidelines established by the Canadian Council on Animal Care.Two-day-old (P2) Sprague Dawley rat pups and their mothers were ordered from Charles River  and acclimatized for 3 days. The rats were housed in standard cages with 1)  and Kineret .Chemicals were purchased from the following manufacturers: human rIL-1β , LPS , rytvela (peptide 2). Cells were serum starved overnight and treated with 100 ng/mL IL-1β for 4 h. Cells were pre-incubated for 30 min with peptides 1, 5, or 6 (10−6M) or Kineret (1.0 mg/mL) prior to the experiments to reach equilibrium (n = 4 each treatment). Cells were harvested and incubated for 5 min in RIBOzol (AMRESCO). RNA was extracted according to manufacturer's protocol and RNA concentration and integrity were measured with a NanoDrop 1,000 spectrophotometer. A total of 500 ng RNA was used to synthetize cDNA using iScript Reverse Transcription SuperMix . Primers were designed using National Center for Biotechnology Information Primer Blast and are shown in IL1β, IL6, and PTGHS2 [Prostaglandin H synthetase 2 or cyclooxygenase-2 (COX-2)]. Data are representative of 3 experiments (each with n = 4 per treatment group).HEK-Blue IL-33/IL-1β cells were purchased from InvivoGen  and used at passages under 15. HEK-Blue cells were cultured in DMEM growth medium supplemented with 10% serum, 50 U/mL penicillin, 50 mg/mL streptomycin, 200 mg/mL zeocin, and 100 mg/mL hygromycin. Cells were grown in regular conditions  or Kineret (1.0 mg/mL) for 30 min, followed by treatment with a constant concentration of IL-1β (100 ng/mL), and then incubated at 37°C for 4 h. Levels of secreted alkaline phosphatase in cell culture supernatant were determined using the QUANTI-Blue assay, according to the manufacturer's instructions (InvivoGen). Alkaline phosphatase activity was assessed by measuring the optical density (OD) at 620–655 nm with a micro plate reader . Data are representative of 3 experiments (each with n = 4 per treatment group).HEK-Blue IL-33/IL-1β cells (InvivoGen) were pretreated with peptides 2) and maintained under passage number 15. Cells were equilibrated with 1, 5, or 6 (10−6M) or Kineret (1.0 mg/mL) for 30 min, after which time they were exposed to IL-1β (100 ng/mL) for 15 min. Cells were harvested and lysed on ice for 30 min using a radioimmunoprecipitation assay buffer  supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and cOmplete™ EDTA-free protease inhibitor cocktail . Protein concentrations were determined using a Bradford protein assay (Bio-Rad) on 96-well plates with a microplate reader (EnVision Multilabel reader) measuring OD at 595 mm. Bovine serum albumin serial dilutions were used to generate a standard curve. Lysates were then mixed with 4X reducing sample buffer (Bio-Rad).RAW Blue cells (Invivogen) were grown under standard conditions  in a 5% acrylamide stacking gel, and samples were electrophoresed in a 12% acrylamide resolving gel for 1.5 h at 120 V, followed by a 1 h transfer onto polyvinylidene difluoride (PDVF) membranes at 100 V. Membranes were blocked and incubated with 1:1,000 dilution of primary antibody and 1:20,000 dilution of secondary antibodies according to the manufacturer's instructions. Antibodies used were for phospho-p38 MAPK , p38 MAPK , SAPK/JNK , phospho-SAPK/JNK , ROCK2 (Thermo Fisher Scientific PA5-21131), phospho-ROCK2 (Thermo Fisher Scientific PA5-34895), and goat anti-rabbit conjugated to horseradish peroxidase . Membranes were imaged using an Amersham Imager 600  using Clarity Western ECL Substrate (Bio-Rad). The intensity of protein bands was quantified using ImageJ and standardized using total (phosphorylated + non-phosphorylated) protein content. Data are representative of three independent experiments.1 was performed as described previously   and 1 mg/kg was injected 12 h after stimulation (n = 4 each treatment). Mice delivery was assessed every hour until term (G19–G19.5). A mouse is considered as delivering prematurely if the first pup is delivered earlier than G18.5.Timed-pregnant CD-1 mice at 16.5 days of gestation (G16.5) were anesthetized with 2% isoflurane and received an intraperitoneal injection of LPS . To control for the effects of hyperoxia on the lactation of the dams, dams of hyperoxic litters were switched with dams of control litters on P8.On P5, litters were transferred to a controlled hyperoxic environment (Biospherix OxyCycler A84XOV) and maintained at 80 ± 1% O1, 5, or 6 or 3 mg/kg/day of Kineret from P5 to P10. These doses were determined from our previous study  vehicle, 2 mg/kg/day of peptides On P10, pups were euthanized by decapitation under 2% isoflurane anesthesia. Eyes were enucleated and fixed in 4% paraformaldehyde for 1 h at room temperature before washing twice with PBS and storage at 4°C in PBS until further processing.Under a dissecting microscope, the cornea and lens were gently removed from the eyes and remnants of the hyaloid vasculature were removed from the retinas using surgical scissors and tweezers. The retinas were gently removed from the underlying sclera/choroid complex.2, 1% Triton X-100, 1% TRITC-conjugated lectin cell endothelial marker from Bandeiraea simplicifolia  and a 1:500 dilution of rabbit anti-ionized calcium-binding adapter molecule (Iba)-1 antibody  in PBS. The retinas were then washed thrice with PBS and incubated with a secondary antibody solution consisting of 1% BSA, 0.1% Triton X-100, 0.05% Tween-20 and 1:500 Alexa-594-conjugated donkey anti-rabbit IgG  in PBS for 2 h at room temperature. Retinas were washed thrice with PBS and then flat-mounted onto glass slides with coverslips and Fluoro-Gel mounting medium .Retinas were treated at room temperature for 1 h with a blocking solution consisting of 1% bovine serum albumin (BSA), 1% normal goat serum, 0.1% Triton X-100 and 0.05% Tween-20 in PBS. The retinas were then double-labeled overnight at 4°C with gentle shaking in an antibody solution consisting of 1 mM CaClFor assessment of vaso-obliteration, retinas were imaged using an epifluorescence microscope at 10X magnification (Zeiss AxioImager Z2) and version 4.8 of the AxioVision software. Images were captured and stitched together using the software's MosaiX feature. Iba-1 staining was assessed with the same microscope at 20X magnification, and a total of 4 fields per retina were imaged halfway between the optic nerve and the edge of the retina.Representative images of Iba-1 staining were taken using a laser scanning confocal microscope (Olympus IX81 with Fluoview FV1000 Scanhead) with the Fluoview Software at 30X magnification.The FIJI software was used to the quantify the area of vaso-obliteration in each retina, expressed as a percentage of the area of the whole retina. The number of Iba-1-positive cells was counted using the cell counter plug-in in FIJI, and the average of cell counts in 4 fields per retina was calculated.p was < 0.05 and expressed as mean ± SEM.Data was analyzed using GraphPad Prism 7 with one-way ANOVA and Dunnett's test for multiple comparisons. Outliers were detected using Grubb's test. Results were treated as significant when -, ψ-, and ω-dihedral angles such that Agl and Hgl residues prefer to situate at the i+1 residue in β-turn sequences contingent on sequence stereochemistry  were performed by approaches featuring preparation of the N-Fmoc-dipeptides in solution, followed by their incorporation into the final peptides using solid-phase peptide synthesis. Although effective means for introducing Agl and Hgl residues directly on solid phase have been developed -Agl-r)-7, N-Boc-d-methionine 8 was coupled to tert-butyl r)-Agl-(R)-Val-Ot-Bu 11 in 58% yield - and -18 were prepared from N-Fmoc-d- and l-methionine methyl esters (R- and S-15) using a modification of the reported three-step protocol in which N-Fmoc-vinylglycine methyl ester 17 was synthesized by a flow process featuring elimination of N-Fmoc-methionine sulfoxide methyl ester 16 using 2,4-dichlorotoluene at 200°C, and epoxidation of olefin 17 was performed by microwave irradiation using m-CPBA in toluene at 80°C -18 was reacted with tert-butyl (R)-valinate 9 using a catalytic amount of benzoic acid and trifluoroethanol under microwave irradiation at 80°C for 90 min to provide N-Fmoc--Hgl-d-Val-Ot-Bu [-19] in 89% yield. A Mitsunobu approach was employed for the synthesis of the cis-Hgl isomers by inversion of the alcohol stereochemistry of their trans-counterparts - and -diasteromers of epoxide 18 and enantiomers of valine 9, the trans- and cis-Hgl dipeptide diasteromers 12 were, respectively, synthesized in 84 and 83% yields.The synthesis of Hgl peptides in solution and on solid phase has been accomplished using oxiranyl glycine  were performed using standard Fmoc-based solid-phase synthesis on Rink amide resin  spectroscopy of peptides 1, 5, and 6 in water were first measured to begin examining the influences of Agl-Val and Hgl-Val dipeptide configuration on conformation. The conformers in water likely represent the geometry that initially binds the receptor. Restraint that pre-organizes a favorable binding conformer may facilitate molecular recognition by diminishing the entropy costs for folding.Understanding the relationships between the preferred conformations of peptides 1 exhibited a curve shape characteristic of a disordered random coil structure -Agl3]-, [-Hgl3]- and [-Hgl3]-1, all exhibited negative and positive maximum that were, respectively, observed at 198–207 and 221–227 nm, indicative of a β-turn conformation (S)-Agl3-(S)-Val4]-, [-Hgl3-(S)-Val4]- and -1 gave similar but inverted curve shapes typical of β-turn conformers with notably greater ellipticity -Hgl3-(S)-Val4]-1 exhibited a curve shape indicative of a β-turn conformer -Hgl3]-1 examined in trifluoroethanol (TFE), MeOH and hexafluoroisopropanol (HFIP), and the greatest ellipticity was seen in 5% TFE in water.The CD spectrum of the parent peptide tructure . Contingormation . Inversi1 and derivatives were ascertained in vitro in RAW-blue and HEK-blue cells which were stimulated with IL-1β. The QUANTI-blue assay was employed to measure concentrations of secreted alkaline phosphatase, a reporter product from the transcription of the NF-κB gene. No peptides that were tested exhibited any noticeable inhibition of NF-κB signaling , p38 mitogen-activated protein kinases (p-38) and Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) in RAW-blue cells, after pre-incubation with peptides 1, 5, or 6 and stimulation with IL-1β (- than s)-Hgl3]- and [(3R)-Agl3s)-Val4--Agl3]-, [(3R)-Agl3]-, [-Hgl3]- and - and [(3S)-Agl3-(S)-Val4]-1 and recovered in part in certain Hgl3-(S)-Val4 analogs with [-Hgl3-(S)-Val4]-1 exhibiting ability to inhibit the expression of all three genes.The effects of peptides  peptide . On the 1 is known to bind to IL-1R -Hgl3]-1, which exhibited a significantly lower (p = 0.0011) ability to compete with radiolabeled peptide 1 -Val peptides 5 and 6 exhibited little efficacy.The spectrum of rth PTB, , featuring labor . On the 1 and a subset of analogs 5 and 6 that were previously tested in the PTB model: e.g., [(3S)-Agl3]-, [(3R)-Agl3]-, [-Hgl3]-, and [-Hgl3]-1 which exhibited the best activity, -1 which had partial efficacy and -1, which was inactive in delaying labor.A model of OIR was next used to examine peptide 1 and Kineret both diminished the extent of vaso-obliteration to 15–20%  in the PTB model, demonstrated efficacy in reducing vaso-obliteration to a somewhat lesser extent than peptide 1. On the other hand, [-Hgl3-(S)-Val4]-1, which had no effect in the PTB model, was also ineffective in curbing vaso-obliteration.Exposure of rat pups to 80% oxygen from days 5 to 10 of life resulted usually in vaso-obliteration of ~35% of the retinal capillaries, extending radially from the optic nerve . Peptideo 15–20% . Among t1 -Hgl3]-, [-Hgl3]- and [-Hgl3]-1 prevented partially the activation of microglia; [(3S)-Agl3]-, [(3R)-Agl3]- and [-Hgl3-(S)-Val4]-1 had no appreciable effect on microglial morphology (S)-Agl3]-1, which modestly (< 20%) reduced microglial density despite not influencing microglial morphology. Peptides 1, 5, and 6, which prevented microglia activation, caused a statistically significant reduction in vaso-obliteration area. In summary, certain [Hgl3]-1 analogs behaved like the parent peptide and exhibited protection against vaso-obliteration in the hyperoxic phase of OIR, due in part to mitigation of microglial activation.Immuno-histochemical staining for Iba-1 was used to assess microglial activation and density, because microglia have been shown to be mediators of vaso-obliteration in the context of OIR  but fail to address the underlying inflammatory processes responsible for labor  and on the transcription of downstream pro-inflammatory genes that are mediated by IL-1β , but did not affect NF-κB signaling. Moreover, peptide 1 delayed significantly PTB in mice induced with LPS and reduced vaso-obliteration in the OIR murine model.Peptide 1 was performed by replacing -Thr3-(R)-Val4 with all four possible Agl3-Val4  and eight possible Hgl3-Val4  diastereomers. Among the twelve analogs of peptide 1, those possessing common backbone stereochemistry  exhibited circular dichroism spectra indicative of β-turn conformers in water: [(3R)-Agl3]-, [-Hgl3]- and [-Hgl3]-, [(3S)-Agl3-(S)-Val4]-, [-Hgl3-(S)-Val]-, [-Hgl3-(S)-Val]-1. Moreover, [-Hgl3-(S)-Val]-1 also exhibited a CD curve indicative of a turn conformer.Conformational constraint of peptide R,4S)-Hgl3]-1 exhibited the most similar activity as the parent peptide in the in vitro and in vivo assays with slightly reduced potency in inhibiting JNK. Moreover, [-Hgl3]-1 was also typically as potent as 1 but had slightly reduced abilities in inhibiting p38 and ROCK2. Although their CD spectra and conformers differed in water, both isomers possess trans-Hgl residues and (R)-Val stereochemistry, indicating the importance of the β-hydroxyl group and gauche-(–) χ-dihedral angle side chain geometry for maximum activity.Among the constrained analogs, [(3R)-Agl3]- and [(3S)-Agl3]-1 correlates with their ability to block JNK without inhibitory potency on p-38 and ROCK2. The importance of the hydroxyl group for activity on the latter kinases and for ability to reduce vaso-obliteration in the OIR model is illustrated by the potency of the corresponding Hgl analogs. Notably, [-Hgl3]-1, which positions the hydroxyl group in a gauche-(+) χ-dihedral angle side chain orientation, maintains some potency on all three kinases—with best activity on ROCK2—and exhibits moderate and strong activities in the PTB and OIR models, respectively.The contrast of high potency and inactivity exhibited in the PTB and OIR models, respectively, by both [-Hgl3]-1, which inhibited strongly ROCK2 but had no effects on JNK and p-38, was inactive in the PTB model. The weaker potency in the PTB assay of the (S)-Val analogs correlated with their lack of inhibitory activity on JNK. Although (R)- instead of (S)-Val may be a prerequisite for binding IL-1R, the latter may also be more susceptible to enzymatic cleavage by proteases -Hgl3]-1, which displaced radio-labeled 1 to a lesser extent despite being the most efficacious molecule in vitro and in vivo , a technique that is invasive and technically demanding  and assisted in synthesis of derivatives. SC and WL supervised the progress of the project, edited, and proofread the manuscript. All authors have read the final manuscript and agree to be accountable for the content of this work.AG wrote the manuscript, synthesized and purified compounds, and conducted circular dichroism analyses. CC wrote the manuscript and conducted The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tadalafil is a cytochrome P450 (CYP) 3A4 substrate. Because there are few data on drug-drug interactions, it is advisable to take sufficient consideration when co-administering tadalafil with CYP3A4 inducers or inhibitors. This study was conducted to assess the effect of ticagrelor, a CYP3A4 inhibitor, on the pharmacokinetic properties of tadalafil after oral administration to rats. A total of 20 Sprague–Dawley male rats were randomly divided into the non-pretreated group and ticagrelor-pretreated group, and tadalafil was orally administered to each group after pretreatment with or without ticagrelor. Blood samples were collected at predetermined time points after oral administration of tadalafil. As a result, systemic exposure of tadalafil in the ticagrelor-pretreated group was significantly increased compared to the non-pretreated group (1.61-fold), and the clearance of tadalafil in the ticagrelor-pretreated group was significantly reduced than the non-pretreated group (37%). The prediction of the drug profile through the one-compartment model could explain the differences of pharmacokinetic properties of tadalafil in the non-pretreated and ticagrelor-pretreated groups. This study suggests that ticagrelor reduces a CYP3A-mediated tadalafil metabolism and that tadalafil and a combination regimen with tadalafil and ticagrelor requires dose control and specific pharmacotherapy. Erectile dysfunction (ED), the most prevalent complaint in males, is the persistent inability to maintain an erection . Various®), the approved PDE5 inhibitor, is used in the treatment of ED and is one of the most frequently prescribed PDE5 inhibitors [Tadalafil , the platelet aggregation inhibitor, belongs to P2Y12 receptor antagonists and is used for the treatment of CAD [12 receptor antagonist [Ticagrelor ) were kindly obtained from Korea United Pharma Inc. . Dimethyl sulfoxide, formic acid, and polyethylene glycol 400 (PEG 400) were purchased from Sigma–Aldrich . Acetonitrile and methanol were purchased from J.T. Baker . Analytical grade reagents were used throughout this study. Overall, distilled water was used.v/v), and the flow rate was 0.4 mL/min. The temperature of the column and autosampler were set as 30 °C and 4 °C, respectively. The positive ion mode using Agilent jet stream electrospray ionization (AJS-ESI) was applied to record the scan mass spectra. The ion transitions of tadalafil and IS were set as 390.4→268.1 m/z and 876.4→308.1 m/z, respectively, and detected with a multiple reaction monitoring (MRM) mode. The collision energies for tadalafil and IS were 10 V and 30 V, respectively. The cell accelerator voltage was 5 V and the dwell time was set as 200 ms. The source parameters were set as follows: Gas temperature 200 °C, gas flow 14 L/min, nebulizer 20 psi, sheath gas heater 250 °C, sheath gas flow 11 L/min, capillary 3000 V, and nozzle voltage 1500 V.The concentrations of tadalafil were analyzed by a liquid chromatography tandem-mass spectrometry (LC-MS/MS) system equipped with Agilent 1290 series and Agilent 6495 Triple Quad LC/MS . A YMC-Triart C18 column  was used. The mobile phase was a mixture of 0.2% formic acid in acetonitrile and 0.2% formic acid in distilled water  at LLOQ and less than 15% CV at all other concentrations) [In this analysis, the most abundant ion transition of tadalafil (390.4→268.1 m/z) was selected to determine the lowest limit of quantification (LLOQ), and the LLOQ of tadalafil was 3 ng/mL. The range of calibration curve of tadalafil was set to 3–6670 ng/mL. The curve was written with a weighted linear regression (1/x) and showed excellent linearity with Rrations) . The acqAll animal experiments were carried out in accordance with the protocol (No. CNU-01167) and the “Guidelines in Use of Animal” approved by Chungnam National University Institutional Animal Care and Use Committee . Male Sprague–Dawley rats  were purchased from Nara-Biotec . All animals were housed in a dark-light cycle of 12 h at 22 °C and were allowed free access to water and food.g for 5 min at 4 °C. The plasma was collected and stored at −20 °C until the concentration of tadalafil was analyzed by LC-MS/MS.The experiment was performed in a parallel design. A total of 20 rats were randomly divided into two groups. The control group, Group N , was orally administered for 7 days only with the vehicle . Group T , an experimental group, received 10 mg/kg of ticagrelor in the vehicle once a day for 7 days orally. Thereafter, on the seventh day, tadalafil (2 mg/kg) was orally administered 30 min after the final administration of the vehicle or ticagrelor. Doses of all samples were calculated according to the weight of the rats and administered using gavage. The dose of ticagrelor and tadalafil given to rats were determined by converting the human doses  to animal doses with body surface area, according to the United States Food and Drug Administration guidelines . Blood (v/v% formic acid) containing 500 ng/mL of IS was added to 20 µL of the plasma sample. After shaking for 5 min, the mixture was centrifuged at 15,000× g for 5 min. The supernatant (150 µL) was transferred and 5 µL of the sample was injected into the LC-MS/MS.Protein precipitation was applied to extract tadalafil from plasma. Briefly, 200 µL of acetonitrile (0.2 max)) and the time to reach the maximal plasma concentration (Tmax) of tadalafil were determined from the plasma concentration-time profiles. The area under the plasma concentration vs time curve from 0 to 24 h (AUC0–24) was calculated by the linear trapezoidal rule and the area under the plasma concentration vs time curve from 0 h to infinite time (AUC0–∞) was estimated by extrapolating time to infinity. The elimination half-life (T1/2) and apparent total clearance (CL/F) were determined from the ln 2/elimination rate constant and dose/AUC0–∞, respectively [Pharmacokinetic data were analyzed based on the non-compartment analysis of WinNonlin software 8.1 . The maximum plasma concentration of tadalafil .In this model, the shift of the tadalafil amount in the gut compartment and the central compartment is described by the following equations :dGdt= −KpredCon) using the one-compartment model were estimated by the following equation [V/F indicates the central volume of distribution of tadalafil.Predicted plasma concentrations of tadalafil  and the statistical analysis was performed using GraphPad Prism 8 software .Values are represented as mean ± standard deviation (SD). A student’s t-test was applied for statistical significance of differences  and AUC0–∞ . The Cmax of Group T slightly increased 1.15-fold compared to that of Group N, but there was no significant difference (p = 0.3332). The ratio of ACU0–24 and AUC0–∞ between Group N and Group T exceeded the range of 0.8–1.25, where no pre-specified pharmacological effect was observed [The pharmacokinetic profiles of tadalafil for Group N and Group T are shown in observed .max, T1/2, and CL/F showed statistically significant differences between Groups T and Group N. In the case of Tmax, the absorption of tadalafil in Group T was delayed (Tmax of 3.22 ± 1.30 h) compared to that in Group N (Tmax of 1.45 ± 0.50 h) (p < 0.005). Thus, the tadalafil plasma concentration remained high until 8 h. This result indicated that the reduced metabolism of tadalafil by CYP3A inhibition caused the absorption of the drug for a longer period of time [1/2 compared to Group N (p < 0.005) and the CL/F value of Group T was significantly lower than that of Group N (p < 0.005). These results showed that the inhibition of hepatic CYP3A metabolism and the reduction of the first pass effect increased the exposure of tadalafil [As a result of CYP inhibition by ticagrelor, the plasma concentration of tadalafil decreased more slowly after co-administration of ticagrelor and tadalafil than after tadalafil alone. T of time . In partadalafil .0–24, AUC0-∞, Tmax, and half-life of tadalafil. In particular, the half-life of tadalafil increased from 3.15 h in Group N to 4.47 h in Group T, resulting in the increased AUC0–24 (1.61-fold), AUC0–∞ (1.66-fold), and Tmax (2.22-fold). In addition, the result of 37% reduction of tadalafil clearance in Group T compared with Group N supported our findings. These data suggest that ticagrelor inhibits tadalafil metabolism due to drug-drug interaction with tadalafil.In general, the non-compartment analysis parameters of tadalafil showed statistically significant differences between Group N and Group T . In the The one-compartment model was successfully applied to describe each group in terms of the plasma concentration-time profile after a single oral administration of 2 mg/kg tadalafil. Among the various compartment models, the one-compartment model, which is the simplest and best suited to the observed pharmacokinetic profile of tadalafil, was used to compare the observed pharmacokinetic profile with the fitted pharmacokinetic profile. The one-compartment modeling supports the results of the non-compartment analysis that changed by the co-administration of ticagrelor. The modeling-based comparison allows for greater confidence in the metabolic inhibition effect by comparing the observed pharmacokinetic profile with the fitted pharmacokinetic profile, as well as by matching with the parameters of the non-compartment analysis. Additionally, the model-based approach was used to evaluate a more specific and quantified absorption, elimination, and distribution of tadalafil in vivo.As shown in e and V/F were decreased significantly in Group T compared to Group N. The Ke of Group T (0.13 ± 0.03 h−1) were decreased compared with that of Group N (0.17 ± 0.05 h−1) (p < 0.05). Moreover, the V/F of Group T (3.36 ± 0.95 L/kg) was significantly decreased compared with that of Group N (4.39 ± 0.78 L/kg) (p < 0.05). These reductions led to a decrease in the clearance of tadalafil. In addition, the Ka of Group T was also decreased, but there was no statistical significance (p = 0.1933)  . HoweverIn addition, the results from the modeling indicate that ticagrelor is a weak CYP3A inhibitor. The weak inhibitor is defined as a substance that increases the AUC value of the CYP substrate by 1.25-fold to 2-fold, or that it reduces the clearance of the CYP substrate by 20–50%, and ticagrelor is also classified as this inhibitor ,41. ChanIn a clinical aspect, tadalafil shows robust safety and tolerability . HoweverIn our study, the plasma concentration-time profile of tadalafil was significantly changed by co-administration with ticagrelor. A ticagrelor-inhibited CYP3A-mediated tadalafil metabolism and the systemic exposure of tadalafil increased by pretreatment with ticagrelor. Co-administration of tadalafil with ticagrelor increased the AUC of tadalafil by approximately 61% and decreased the clearance of tadalafil by 37%. These results suggest that the co-administration of tadalafil and ticagrelor may need dose control and specific drug therapy to avoid side effects from drug-drug interactions. Therefore, CAD patients receiving ticagrelor should be closely monitored when administering tadalafil concomitantly. For further studies, an additional experiment is expected to identify the variation of clinical efficacy of tadalafil by co-administration with ticagrelor, a CYP3A4 inhibitor.


Marchantia emarginata and has been functionally characterized. Experiments based on recombinant protein showed that the enzyme uses ω-hydroxy fatty acids or primary alcohols as its acyl acceptor and various hydroxycinnamoyl-CoAs—preferentially feruloyl-CoA and caffeoyl-CoA—as acyl donors at least in vitro. The transient expression of a MeHFT-GFP fusion transgene in the Nicotiana benthamiana leaf demonstrated that MeHFT is directed to the cytoplasm, suggesting that the feruloylation of cutin monomers takes place there. The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle. The main constituent of the cuticle is the lipid polyester cutin, which is composed of aliphatic and aromatic domains. The aliphatic component is a polyester between fatty acid/alcohol and hydroxycinnamoyl acid. The BAHD/HxxxD family enzymes are central to the synthesis of these polyesters. The nature of this class of enzymes in bryophytes has not been explored to date. Here, a gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (HFT) has been isolated from the liverwort  Theglycerol ,5,6. Altglycerol .Arabidopsis thaliana BAHD/HXXXD family acyltransferase, encoded by the gene At5g41040, has been shown to have HHT activity [At5g41040 reduces both the ferulate ester and ω-hydroxy fatty acid contents of suberin, resulting in the development of a thickened peel and an increased rate of water loss [A. thaliana mutant lacking cutin feruloyltransferase (a distant homolog of HHT1) activity displays no marked alteration in its cuticle function in relation to ion permeability, water loss or pathogen infection [The enzymes responsible for acylation belong to the large BAHD/HXXXD acyltransferase family , almost activity ,21 and iter loss . In contnfection . Populus trachocarpa FHT1 enzyme accepts both p-coumaroyl and feruloyl CoA as a thioester donor and is able to acylate ω-hydroxy acids and fatty alcohols in vitro. Heterologously expressing the Populus gene in A. thaliana has the effect of increasing, by as much as 45%, the incorporation of phenolic esters into both the leaf cutin and the root and seed suberin; the accumulation of ferulate esters conferred thereby confer a significantly increased tolerance to salinity stress [The y stress . WoLF PSy stress  has indiy stress .M. emarginata (MeHFT) has been characterized. The enzyme appears to be involved in the formation of alkyl hydroxycinnamate esters and is active in the cytoplasm; in vitro, the enzyme can use either feruloyl or caffeoyl-CoA as its acyl donor, and ω-hydroxyacids or fatty alcohols as its acyl acceptor. Bryophytes, the group of non-vascular plants which includes mosses, liverworts and hornworts, were among the first plants to colonize land. To achieve this, they needed to evolve an number of adaptations to cope with a range of abiotic stresses which are not relevant in a marine or aquatic environment . The livM. emarginata transcriptome (SRP078649), a sequence encoding a putative HFT was identified and given the designation MeHFT. The sequence’s length was 1413 nt and its predicted product a 470 residue protein of molecular mass 51.93 kDa and pI 4.91. The predicted polypeptide’s sequence shared, respectively, 50.6%, 48.2%, 42.3%, 44.3% and 49.5% identity with those of PtFHT , AtHHT1X515962: ,21), AtDX515962: ), AtFACTX515962: ) and StFX515962: ). Sequenp-coumaroyl CoA, caffeoyl CoA and feruloyl CoA) and acyl acceptors  showed that when recombinant MeHFT was provided with feruloyl CoA and 1-dodecanol, a single prominent HPLC peak was observed  , which iobserved A. MS/MS with C12 B. The pr plasmid A. When Croyl CoA . The datroyl CoA  showed tcat/Km ratios in the presence of 1-decanol (3181 M−1s−1) and 1-dodecanol (3015 M−1s−1)  . When prr plants . These m-decanol 81 M−1s−135S::MeHFT-GFP trangene in the N. benthamiana leaf is shown in The pattern of expression of the pM. emarginata thallus was raised under a 12 h photoperiod and a constant temperature of 25 °C. Harvested thalli were snap-frozen in liquid nitrogen and stored at −80 °C. Nicotiana benthamiana plants were soil-grown for 5–6 weeks under a 12 h photoperiod, with a day/night temperature regime of 24 °C/22 °C. p-coumaroyl CoA and caffeoyl CoA were synthesized from ferulic acid, p-coumaric acid and caffeic acid, respectively, using liverwort 4-coumarate CoA ligase [All chemicals and reagents required were purchased from either Sigma–Aldrich  or Energy Chemical , unless otherwise noted. Feruloyl CoA, A ligase , then puA ligase . The proMeHFT sequence and certain selected related plant proteins was carried out using routines implemented in MEGA v5.1 software. [A phylogenetic analysis involving the oftware. . The NeiM. emarginata thallus was extracted using a CTAB-based method [MeHFT was amplified from a cDNA template using PrimeSTAR HS DNA polymerase (Takara) driven by the primer pair MeHFT-F/-R , and ligated into the corresponding cleavage sites of pET32a  in order to generate a transgene which encoded a His-tagged version of MeHFT. To heterologously express this transgene, it was transferred into competent E. coli BL21 (DE3) cells Expression of the transgene was induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside to the culture medium, and the cells were held at 16 °C for 16 h. Recombinant protein was purified from the medium by passage through a Ni-NTA Sefinose His-bind column , following Gao et al. [Total RNA from two month old d method , and revo et al. . The cono et al. .p-coumaroyl-CoA, caffeoyl-CoA or feruloyl-CoA and 20 μM acyl acceptor (see below) in 0.1 M Tris-HCl buffer, pH 7.5. The reactions were initiated by the addition of the recombinant protein, held at 40 °C for 20 min, and terminated by the addition of 50 μL acetonitrile. After centrifugation  to remove any precipitated protein, a 20 μL aliquot of the mixture was injected into an HPLC device equipped with C18 reverse-phase column . The material was eluted by passing a mixture of 0.2% v/v glacial acetic acid in water (A) and acetonitrile (B) provided at a flow of 0.8 mL min−1. In the period 0–5 min, the eluate was composed of 85% A, 15% B; from 5 to 25 min, the proportion of B was raised linearly to 100%; from 25 to 35 min, the eluate was 100% B; and from 35 to 40 min, the proportion of B was lowered linearly to 15%. UV absorption was monitored at 330 nm.Each 50 μL enzymatic assay reaction comprised 1 μg recombinant protein, along with 50 μM of either www.graphpad.com). The quantity of reaction product was calculated based on a standard calibration curve. The reaction kinetics of the recombinant MeHFT was tested in two sets of experiments. In the first, the recombinant enzyme was provided with 0.1 mM 1-decanol and a range of concentrations of feruloyl-CoA in 0.1 M Tris-HCl buffer (pH 7.5), while in the second, the feruloyl-CoA concentration was fixed at 0.1 mM and the 1-decanol concentration was varied. Both sets of experiment utilized 1 μg purified protein, and were held at 40 °C for 10 min. Kinetic data were calculated on the basis of the Michaelis-Menten equation, using Graphpad Prism 5 software (MeHFT sequence was inserted into pGWB5 (p35S::GL2-GFP) (www.thermofisher.com) for the purpose of determining the subcellular localization of MeHFT. After its amplification driven by primer pair attB1-MeHFT-F/R  and held at 28 °C until their OD600 reached about 0.5. The cells were then harvested by centrifugation , rinsed in 50 mM MES/KOH (pH 5.6), 2 mM Na3PO4, 28 mM glucose, 100 mM acetosyringone, then re-suspended in the same buffer to an OD600 of 1.0. The resuspended cells and p19 were mixed in a 1:1 ratio and the mixture incubated for 3 h at 25 °C, then infiltrated with a syringe into the base of a N. benthamiana leaf [The eHFT-F/R , the seqana leaf . After tM. emarginata may reveal new insights into the diversity of acyltransferases.This represents the first report of the in vitro functionality of a bryophyte HFT. The identification of a member of the BAHD family in as basal a land plant as the liverwort


Trauma informed care (TIC) is a whole system organisational change process which emerged from the seminal Adverse Childhood Experiences (ACE) study, establishing a strong graded relationship between the number of childhood adversities experienced and a range of negative outcomes across multiple domains over the life course. To date, there has been no systematic review of organisation-wide implementation initiatives in the child welfare system. As part of a wider cross-system rapid evidence review of the trauma-informed implementation literature using systematic search, screening and review procedures, twenty-one papers reporting on trauma-informed implementation in the child welfare system at state/regional and organisational/agency levels were identified. This paper presents a narrative synthesis of the various implementation strategies and components used across child welfare initiatives, with associated evidence of effectiveness. Training was the TIC implementation component most frequently evaluated with all studies reporting positive impact on staff knowledge, skills and/or confidence. The development of trauma-informed screening processes, and evidence-based treatments/trauma focused services, where evaluated, all produced positive results. Whilst weaknesses in study design often limited generalisability, there was preliminary evidence for the efficacy of trauma-informed approaches in improving the mental and emotional well-being of children served by community-based child welfare services, as well as their potential for reducing caregiver stress and improving placement stability.  Trauma informed care (TIC) is a whole system organisational change process which seeks to embed theoretically coherent models of practice across diverse settings and roles, including child welfare, family support, justice, mental health and education. It emerged from the findings of the seminal Adverse Childhood Experiences (ACE) study in the U.S.  with subIn recognising the impact of childhood adversity on child and adult outcomes, trauma-informed services strive to build trustworthy collaborative relationships with children and the important adults in their lives, as well as improve consistency and communication across linked organisations and sectors, with the aim of mitigating the impact of adversity by supporting and enhancing child and family capacity for resilience and recovery. TIC also seeks to change organisational practices and reduce the use of some practices, such as restraint or seclusion, which may inadvertently exacerbate the detrimental effects of severe adversity and constrain client/service user engagement. TIC advocates have developed a set of key assumptions and principles to help design responsive, holistic and effective systems of care . These iThe child welfare workforce interfaces with children and adults who have experienced adversity and trauma on an everyday basis. Indeed, it can be argued that no other child-serving system encounters a higher percentage of service users with trauma histories, whether it be in family support, child protection, foster, kinship or residential care. Experiences of maltreatment and neglect, parental mental ill health, domestic violence and substance misuse often co-occur , while rIn an effort to develop more trauma-informed child welfare systems, various national initiatives, practice and training models have emerged. Of particular note is the work of the National Child Traumatic Stress Network (NCTSN) in the United States. Established by Congress in 2000, the NCTSN is a group of 70 treatment and research centres across the United States which has been instrumental in implementing trauma-informed child welfare initiatives in the USA and internationally. The development of trauma-informed practice in child welfare has also seen substantial federal funding with the Administration for Children and Families (ACF), a division of the United States Department of Health and Human Services (HHS), funding five-year demonstration grants in 2011 to develop and evaluate a range of strategies for improving care for children in the child welfare system suffering from exposure to trauma. Strategies included workforce development, trauma screening and referral, dissemination of trauma-focused evidence-based treatments (EBTs), and improved collaboration between child welfare and behavioural health services.Given that TIC requires multiple level change, various guidance and frameworks for implementing trauma-informed approaches in organisations have also been developed. While definitions and terminology vary, Hanson and Lang  identifiHowever, to date, there has been no systematic review of child welfare system initiatives encompassing the broader range of child welfare services outside of residential or out-of-home care. As part of a wider cross-system rapid evidence review of the trauma-informed implementation literature using systematic search, screening and review procedures , this paWhat are the key components of approaches used within systems of care to create trauma-informed practice and what is the evidence of their effectiveness?A systematic search for relevant articles was conducted on the 22nd October 2018 using the databases: International Bibliography of the Social Sciences (IBSS); Science Citation Index Expanded (SCI-EXPANDED)—1970–present); PsycINFO (2002–present); Ovid MEDLINE (ALL 1946 to 31 August 2018); SCOPUS; and ERIC  or different forms of psychotherapy, were excluded, as were those which reported on a specific service/intervention but did not include wider, organisational implementation components.To be eligible for inclusion, papers needed to clearly identify the trauma-informed components of the initiative being implemented and contain some evaluation component with associated data. No limits were placed on the type of outcomes measured and papers could include a range of outcomes such as the impact on service users, improvements to staff knowledge and practice, organisational change, policy development etc.As well as including an evaluation component, articles which only reported on training evaluations also needed to include a pre- and post-test evaluation design to be eligible for inclusion. Literature reviews based on systematic methods were included while non-systematic reviews were excluded, as were studies using a single case design. Additionally, non-English language papers, papers published before 2009, and conference proceedings, dissertations and other papers not published in journals, were excluded. The search identified 5527 potentially relevant articles. References were exported to an excel database and 3824 duplicates removed see . The titThe full text of the remaining 125 articles was then reviewed against the eligibility criteria. Forty-five articles were excluded at this point, primarily because they did not present evaluation data, they only evaluated training with a post-test or qualitative design, or they were not systematic reviews. There was no full-text availability for an additional five papers.The remaining seventy-five papers selected for data extraction contained five systematic reviews which identified definitions and components of TIC relevant to: the juvenile justice system ; out-of-Data extraction entailed extracting key study data  and exporting to an MS Excel spreadsheet. For those papers cited in systematic reviews, the systematic review was the primary source for data extraction supplemented, where necessary, with data from the original article.A narrative approach to synthesis was adopted. Narrative synthesis relies primarily on text to summarise and explain the findings from multiple studies  and is cOf the 75 relevant papers identified through searching the academic literature, 21 papers reported on evaluations of 17 community-based child welfare initiatives involving frontline social workers, family welfare staff and/or other professionals. All originated in the USA. Eight initiatives were large state-wide initiatives usually comprising multiple TIC implementation components and covering multiple professions and agencies, primarily child welfare caseworkers, clinical staff, foster care and adoption services, family preservation services and/or child welfare/treatment facilities. Nine were organisational or agency-level initiatives which generally targeted staff employed in specific agencies such as Child Advocacy Centres, fostering agencies or family preservation services. Across initiatives, service user and training outcomes were the most commonly evaluated elements of the TIC initiatives, followed by evidence-based treatment and trauma-focused services see . EvaluatOnly eight projects reported specifically on outcomes for children and/or their families see . The staSix organisational-level initiatives also evaluated case outcomes. Two initiatives involving child protection/family preservation services reported reductions in child behaviour problems following implementation of the Attachment, Regulation and Competence (ARC) model in a community trauma treatment centre , and incWith the exception of the MCTP outcome evaluation , most stTraining was, by far, the most common component of TIC implementation and was described in almost all of the child welfare papers as a central element of the trauma-informed initiatives under review. Nine of the initiatives specifically evaluated training outcomes, mainly through quantitative pre-test/post-test designs see , althougPost-training follow-up ranged from six weeks to two years, with retention rates between 12% and 89%. Assessment was primarily based on self-reporting, although a number of studies utilised validated measures such as the Evidence-Based Practice Attitudes Scale (EBPAS), the Trauma-Informed System Change Instrument (TISCI) and the Trauma System Readiness Tool ,26,31 toSimilarly, participants in the “Training for Adoption Competency” initiative, which involved 855 professionals employed in mental health, adoption, family service and residential care agencies across 16 States , reporteVarious initiatives stressed the importance of on-going staff support as crucial to maximising the impact of initial training and embedding TIC in practice. Strategies to address this included the use of learning collaboratives ,31,32, cAlthough staff care/self-care features as a key element of various TIC implementation frameworks, specific efforts to address this in community-based child welfare initiatives were limited with the exception of the Connecticut Collaborative on Effective Practices for Trauma (CONCEPT)  and the A number of papers discussed the implementation of specific trauma screening processes, outcomes from training in routine inquiry, or described inclusion of evidence-based screening measures as part of trauma-informed training ,32,34,37In addition to presenting screening data, Lang et al.  noted thIn Michigan Children’s Trauma Assessment Centre , screeniFour state-wide initiatives, the Massachusetts Child Trauma Project , the ArkOther trauma-focused services provided as part of the implementation process included a 24 month, four phase programme to help youth in out-of-home placement achieve permanency and strengthen their connections to supportive adults ; the devMany of the TIC initiatives reported were part of broader, organisation-wide trauma-informed implementation strategies aimed at changing organisational culture and practices. Key elements of implementation therefore focused on targeting leadership buy-in. This was achieved via providing initial training to agency directors and senior management, establishing implementation teams, developing strategic implementation plans and structures, and assessing organisation readiness ,31,32,35The Michigan Children’s Trauma Assessment Centre  placed aA number of papers drew attention to changes made to policies, processes and/or data systems as part of the implementation process ,31,32,37In an effort to embed trauma-informed principles into decision-making processes, the Michigan Children’s Trauma Assessment Centre  developeAlthough not specifically evaluated, a number of other initiatives described efforts to develop trauma-informed policy and procedures. In the CONCEPT initiative in Connecticut , implemeAlthough a number of initiatives reported on steps taken to engage service users as an integral component of implementing TIC in child welfare settings, the data included were largely descriptive with parents and carers the primary targets, rather than children and young people. Initiatives took the form of parent and carer involvement in trauma-informed training ,27,32 anThe KVC Behavioural Healthcare initiative’s implementation of Trauma Systems Therapy (TST) provided one of the most comprehensive examples of a systems-wide training which initially targeted staff and foster parents and later expanded to encompass community partners and birth parents. It was also the only project to present process evaluation data which indicated that, after two years, 67% of foster parents had received trauma-informed training .None of the community-based child welfare projects reported on changes made to offices or other facilities as a means of promoting a positive, safe physical environment in which to engage service users. Previous reviews of the TIC implementation literature have highlighted a variety of methodological difficulties. These include a relative dearth of outcome focused evaluations, particularly in relation to large-scale TIC efforts, a lack of experimental designs, small sample sizes, high attrition rates and short follow-up periods in longitudinal designs ,11,12. IThe review methodology described in this paper also presents its own limitations when interpreting the findings from the child welfare TIC literature. Firstly, it was limited to organisational interventions that were explicitly trauma-informed. Although this allowed for the application of systematic and replicable methods to be applied to evaluate the body of evidence, it excluded interventions that embraced TIC principles without using the language of the trauma-informed. Secondly, as the review was concerned with effective approaches used within systems, it excluded specific trauma-informed clinical interventions and trauma-focused services/interventions which were not delivered as part of a wider programme of organisational change. Thirdly, although systematic search and data extraction methods were applied, this was a rapid evidence review rather than a systematic review and, as such, evaluation of research quality was limited to broad assessment of the study design and reported limitations. Despite these limitations, this review presents preliminary evidence for the efficacy of trauma-informed approaches in improving the mental and emotional well-being of children served by community-based child welfare services, as well as their potential for reducing caregiver stress and improving placement stability. Implementation at the workforce level focused primarily on staff training with all evaluations showing significant increases in staff knowledge, confidence and/or skills in applying TIC principles. These changes were maintained over time, in some cases up to one to two years after initial training. The development of specific TIC standardised measures such as the Trauma-Informed System Change Instrument (TISCI) and the Trauma System Readiness Tool (TSRT) brought additional rigour to these evaluations, enabling researchers/developers to assess baseline levels of knowledge of TIC policy and practice, assess system readiness, identify training and support needs and measure changes over time ,26,31,32Many initiatives also introduced strategies to provide on-going support to staff after initial training through supervision, booster training, coaching and mentoring. While the extent to which these contributed to overall outcomes was unclear, this implementation element was consistently emphasised as central to embedding TIC principles into every-day organisational practice. However, a focus on staff support/self-care was noticeably absent in this review with only two projects detailing specific efforts to address this . To dateIn the domain of trauma-focused treatment/services, the introduction of trauma screening processes was relatively common. Although evaluation was limited and it was not always clear how, or if, this led to changes in the services provided, there was some evidence that this was perceived positively by professionals, resulting in substantial increases in the numbers of children screened at intake by child welfare and mental health professionals ,32. WhilImportantly, a number of state-wide initiatives ,29,31,32At the organisational level, leadership buy-in and strategic planning were the most commonly reported implementation components, although, as with other implementation domains, evaluation was often limited to qualitative findings. Despite these limitations, these elements of TIC implementation were highlighted as essential to success ,32. Top-Other components of organisational change were less prominent in the papers reviewed. Service user engagement formed part of only a few implementation strategies and primarily took the form of parent and carer inclusion in TIC training, although the Massachusetts Child Trauma Project also included service users in leadership teams  and the There is a robust and growing body of research which indicates that severe or chronic adversity in childhood has a significant, negative impact on a child’s development, health and life chances across their life course. Integrating awareness of childhood adversity into the public health agenda and cross-system service delivery  is therefore essential to prevent, recognise and mitigate the impact of trauma on children. This body of research also points to the importance of understanding parental/caregiver histories, who may also be impacted by early adversity. This knowledge has led to the development of models of trauma informed care in diverse practice settings in the USA which seek to mitigate the impact of adversity by promoting collaborative engagement with children and their caregivers and enhance child and family capacity for resilience and recovery. TIC has international reach with country-wide initiatives appearing across the UK and Europe. The rapid evidence review presented here sought, primarily, to explore the evidence pertaining to the organisational change processes required to implement trauma-informed care at a whole systems level within community-based child welfare settings. As part of a wider cross-system rapid evidence review of the trauma-informed implementation literature, twenty-one papers reporting on trauma-informed implementation at state/regional and organisational/agency levels of the child welfare system were identified. The diversity of interventions included in the papers reviewed and a variety of methodological difficulties, intrinsically limit the potential to draw firm conclusions from the literature. Notwithstanding these limitations, a number of multi-component evaluations of large-scale child welfare initiatives have begun to elucidate the positive impact of different TIC components. Training was the component of implementation most frequently evaluated with all studies reporting positive impact in terms of staff knowledge, skills and/or confidence. Other implementation components such as leadership and strategic planning, the development of evidence-based treatment and trauma-focused services, the provision of on-going staff support and the development of trauma-aligned policies and procedures, were also evidenced to varying degrees across many initiatives. The development of trauma-informed screening processes, and evidence-based treatments/trauma-focused services, where evaluated, all produced positive results. Staff support, service user engagement and changes to the physical environment were less prominent in the papers reviewed, pointing to evident research gaps. Whilst weaknesses in study design often limited generalisability, there was preliminary evidence for the efficacy of trauma-informed approaches in improving the mental and emotional well-being of children served by community-based child welfare services, as well as potential for reducing caregiver stress and improving placement stability. This body of literature, with good practice examples embedded within, will assist others in taking forward the important challenge of evidencing the effectiveness of TIC in child welfare settings.


Mouse models have delivered variable recapitulation of Kyasanur Forest disease (KFD) pathology and consistently demonstrated neurological involvement which may be a limited feature of human disease. With the purpose of more accurately modelling human disease progression we infected several small-mammalian models: guinea pigs, hamsters and ferrets with a titered infectious dose of Kyasanur Forest disease virus (KFDV). Clinical indicators of disease severity were observed for seventeen days, on day eighteen a visual post-mortem analysis of visceral organs was conducted. Viral load in selected tissues was measured to infer disease signs and the establishment of viral replication.Daily monitoring did not reveal any observable signs of illness; weight loss was minimal across species and gross pathology did not indicate severe viral infection. Tissue specific tropism and establishment of viral infection was monitored by quantitative real-time polymerase chain reaction (qRT-PCR). No viral replication was detected in ferrets (n = 0/3), but was present in the spleen of guinea pigs (n = 3/3) and the brain of hamsters (n = 3/3). Low levels of viral RNA were detected in multiple hamster tissues  suggesting the possibility of viral tropism and possible adaptation to the host. No serological tests were performed. Cavia porcellus), hamster (Mesocricetus auratus) and ferrets (Mustela putorius furo)). Animals challenged with an infectious dose of Kyasanur Forest disease virus (KFDV) were monitored daily for visual signs of disease, febrility (ferret only), lethality, and weight loss. Post-mortem examination was performed to collect tissue from the: brain, kidney, liver, lung, spleen of each animal. Viral RNA was extracted from tissue samples and quantified by real-time RT-PCR. Presence of viral RNA was interpreted as evidence of viral tropism and replication. We hypothesized that one of the selected non-mouse models may act as a better model for human disease of KFDV infection than current mouse models.In humans, Kyasanur Forest disease (KFD) is described as a biphasic illness that presents with flu-like symptoms in the first stage and may advance to a second stage with hemorrhagic fever manifestations and/or neurological abnormalities , 2. EpidKFDV isolate P9605  was prev4 TCID50 units of passage-two KFDV was intra-peritoneal-injected. The challenge dose was back titrated on Vero E6 cells. Observations of visual disease, weight loss and lethality were conducted for 16 days (hamsters and guinea pigs) or 17 days (ferrets). For ferrets, temperature was also recorded via the subcutaneous IPTT-300 sensors.Each animal species was housed separately in groups of three per cage: female (28–70 days old) outbred LVG Golden Syrian hamsters , female (26–58 weeks old) outbred Hartley guinea pigs  and female (650–800 g) specific pathogen-free (SPF) ferrets  with subcutaneous implanted IPTT-300 temperature and ID transponder devices . The procedures described herein, were approved by the Canadian Science Centre for Human and Animal Health-Animal Care Committee  adhering to the Canadian Council on Animal Care guidelines. Animals were fed and monitored daily for a 7-day acclimation period and for the duration of the experiment. Pre-challenge, each animal was anesthetized with 5% isoflurane in oxygen and 0.1 mL with 6.3 × 10® 480 RNA Master Hydrolysis Probes and a previously published primer design [At the end of the observation period, animals were anesthetized with 5% isoflurane in oxygen and blood was collected. While under anesthesia, animals were euthanized  for gross pathology and tissue collection. Isolated tissues  were inactivated with RLT and viral RNA was extracted with the RNeasy Plus Mini kit . Viral RNA was detected via quantitative real-time polymerase chain reaction (qRT-PCR) using the Roche LightCycler r design , 12.Throughout the course of observation, clinical signs of disease  were not observed and all animals gained weight . TemperaThis pilot study contains several limitations including: small sample size, no replication for statistical inference, single dose-inoculation, and the presence of infectious virus cannot be directly inferred from the detection of nucleic acid . Additio


Enhanced Recovery After Surgery (ERAS) is a multimodal perioperative care bundle aimed at the early recovery of patients. Well accepted in gastric and pelvic surgeries, there is minimal evidence in neurosurgery and neurocritical care barring spinal surgeries. We wished to compare the length of intensive care unit (ICU) or high dependency unit (HDU) stay of patients undergoing elective craniotomy for supratentorial neurosurgery: ERAS protocol versus routine care. The secondary objective was to compare the postoperative pain scores, opioid use, glycemic control, and the duration of postoperative hospital stay between the two groups.In this pragmatic non-randomized controlled trial (CTRI/2017/07/015451), consenting adult patients scheduled for elective supratentorial intracranial tumor excision were enrolled prospectively after institutional ethical clearance and consent. Elements-of-care in the ERAS group were- Preoperative –family education, complex-carbohydrate drink, flupiritine; Intraoperative – scalp blocks, limited opioids, rigorous fluid and temperature regulation; Postoperative- flupiritine, early mobilization, removal of catheters, and initiation of feeds. Apart from these, all perioperative protocols and management strategies were similar between groups. The two groups were compared with regards to the length of ICU stay, pain scores in ICU, opioid requirement, glycemic control, and hospital stay duration. The decision for discharge from ICU and hospital, data collection, and analysis was by independent assessors blind to the patient group.p = 0.04, 0.001, 0.004 respectively). The total hospital stay was similar in both groups.Seventy patients were enrolled. Baseline demographics – age, sex, tumor volume, and comorbidities were comparable between the groups. The proportion of patients staying in the ICU for less than 48 h after surgery, the cumulative insulin requirement, and the episodes of VAS scores > 4 in the first 48 h after surgery was significantly less in the ERAS group – 40.6% vs. 65.7%, 0.6 (±2.5) units vs. 3.6 (±8.1) units, and one vs. ten episodes (The study demonstrated a significant reduction in the proportion of patients requiring ICU/ HDU stay > 48 h. Better pain and glycemic control in the postoperative period may have contributed to a decreased stay. More extensive randomized studies may be designed to confirm these results.CTRI/2018/04/013247), registered retrospectively on April 2018.Clinical Trial Registry of India ( Enhanced Recovery After Surgery (ERAS) is a multimodal perioperative care pathway that has led to a dramatic change in the conventional surgical doctrine . Its impERAS protocols target perioperative stress response with specific goal-directed evidence-based practices . Many suWe describe the use of a modified, multidisciplinary ERAS protocol for neurosurgical patients. This study aims to prospectively analyze the effect of the modified ERAS protocol on the outcomes of patients undergoing elective supratentorial tumor surgeries.Our study was a prospective non-randomized assessor-blinded trial conducted in an 800 bedded tertiary center  as the doctoral thesis requirement of A.E. The institute has a dedicated neuro ICU with 24-h coverage by a dedicated team of doctors and nurses. At the time of this study, the area had a combination of 8 ICU and high dependency beds (HDU), and henceforth we use the term ICU to refer to any of these bed types. The study was granted permission by the institute ethics committee (IEC) and was prospectively registered (CTRI/2017/07/015451). Informed written consent was obtained from all patients. A change in the study design (as suggested by the IEC) lead to retrospective re-registration (CTRI/2018/04/013247).All adult patients of ASA physical status I and II over 18 years (inclusive), with a single supratentorial space-occupying lesion posted for elective craniotomies, were included. Moribund patients requiring emergency craniotomies, uncontrolled diabetics, severely cognitively impaired patients unable to follow simple instructions, and those who did not consent were excluded. Informed consents were obtained, and cohort assignments were based on the patients’ and family members’ agreement to be assigned to the ERAS protocol versus control.An ERAS protocol bundle, based on the existing literature and that proposed by Hagen et al., was agreed upon by the Department of Anesthesiology and Critical care and the Department of Neurosurgery in conjunction with the Critical care nursing and dietetics divisions and approved by the institutional ethics committee . The protocol consisted of primarily three segments – Preoperative, intra-operative, and postoperative. , short-acting opioids (fentanyl – 2 mcg/kg body weight), vecuronium (0.1 mg/kg body weight), and isoflurane titrated to minimum alveolar concentration (MAC) of 1.0. Incision site infiltration with lignocaine 2% (10 mL), invasive arterial pressure monitoring with arterial blood gases on demand, point of care sugar monitoring, pulse pressure variation, and temperature management were available to all anesthetists at all times, but their use was not enforced.Care in the ICU- Standard nurse-led analgesia and sedation management in the ICU followed second hourly documentation of Visual Analogue of pain (VAS) and Richmond Agitation Sedation Scales (RASS), aiming for a VAS < 4 and RASS between − 2 to + 1. Intravenous paracetamol  followed by non-steroidal agents (NSAIDs) as required was administered. Fentanyl (1 mcg/kg body weight) was used only as rescue analgesia for VAS > 4. Dexmedetomidine (first choice) or propofol infusions were used for postoperative sedation of ventilated patients, midazolam being reserved for hemodynamically unstable ones. The unit has standard SOPs for insulin infusion to maintain blood sugars in the 110–180 mg% range. Patients whose blood glucose levels were above 180 mg %were started on an insulin infusion with second-hourly monitoring to reach the target range. Patients meeting pre-set criteria were extubated by the on-floor ICU team of doctors and nurses. However, starting enteral feeds, removing drains, urinary catheters, and ambulation awaited the ICU and neurosurgery consultant rounds the day after the surgery, as per unit protocol.The preoperative ERAS bundle began in Group ERAS (GrE) with structured preoperative counseling and education. The patients and next of kin were informed about the elements of care of the multimodal ERAS protocol. An active patient and caregiver participation was encouraged to improve compliance. All patients received a preoperative complex carbohydrate maltodextrin drink (Preload®) 100 g in 200 mL of clear water the night before surgery and repeated 50 g in 100 mL water 2 h preceding the surgery . Pre-empThe discharge decision from the ICU was taken by the neurosurgical and critical care teams together. Discharge criteria were the same for both groups and included adequate pain control, afebrile state, cardiopulmonary stability, and being able to sit out of bed. Both subsets of patients were followed up till the day of discharge from the hospital.To maximize adherence, an ERAS checklist was attached to all GrE patient-files after obtaining consent. Before starting the study, a workshop was organized to train the nursing teams of both intensive care units and neurosurgery ward to familiarize the staff and resident doctors with the various elements of the ERAS protocol- this module was repeated twice during the study period to cater to changes in staff- mix.Duration of ICU stay defined as calendar days from ICU admission was the primary outcome. The secondary measures were total episodes of the visual analogue score (VAS) > 4, insulin (units) and fentanyl (micrograms) administered in the first 48 h of ICU stay, and the total duration of hospital stay after surgery.All the interventions related to implementing the ERAS protocol were made by the research team belonging to anesthesia , Critical Care (S.T.), and Neurosurgery (R.N.S). Members of the study team were not involved in the decision to discharge from the ICU or hospital.All assessments and documentation  were made by ICU nurses not a part of the study team. After discharge from the ICU, the ERAS checklist was removed from the patient-files, and a blinded assessor collected the data for analysis. The data analyzer was unaware of the group allocation of the patients.The sample size calculation was based on data obtained from the previous six months’ medical records. Seventy percent of patients operated for supratentorial tumors by the same surgery, and the anesthesia team had a postoperative ICU stay of > 48 h. It was hypothesized that the ERAS protocol could bring this down by 50%. For 80% power, an alpha error of 5, and 10% attrition, a sample of 70 patients would be needed.Descriptive statistics were used to compare the patient baseline characteristics. All continuous data which were normally distributed were analyzed using Student’s t-test, whereas nonparametric data were analyzed using the Mann – Whitney U test. A chi-square test or Fisher’s exact test was used for qualitative variables. All the statistical tests were performed using the SPSS software, version 25.A total of 108 patients were eligible, of which 14 patients were excluded for lack of consent. Twenty-four patients (11 in Gr E and 13 in Gr C) were excluded after consent due to logistic reasons such as operation theatre unavailability on the day of surgery. Patients were recruited between August 2017 to October 2018, and the follow-up was completed by the end of November 2018. Figure The baseline demographic characteristics were similar between the two groups Table . The medX2  = 8.571, p = 0.003) The absolute risk reduction was 25.02% , which showed that one out of every four patients benefitted from the ERAS protocol with a reduced ICU stay duration.Number of patients staying in ICU/HDU for more than 48 h was significantly lesser in the ERAS group of patients than in the Control group.  had their urinary catheters removed on the first postoperative day against 13 in Gr C. Of the 14 in Gr E whose catheters were not removed, 9 had SIADH.U = 486.00, p = 0.03, r = .25).There was a significant difference in the insulin administered to the groups - Gr C required a higher cumulative dosage of insulin within the first 48 h after surgery  than the ERAS group   = 9.79, p = 0.02).Postoperative pain scores showed a significant difference between the groups. Twenty-eight out of 35 patients in ERAS did not have even one episode of VAS above 4 in the first 48 h of ICU stay compared to 19 in the control group. .The cumulative fentanyl dose in ERAS group , was significantly lesser as compared to control group   and 1 for abscess- GrE); 1 patient developed an infarct (GrC), two patients developed postoperative cerebral edema (GrC), 1 developed transient renal failure (GrE), and 1 went into septic shock (GrE). The ERAS group of patients did not exhibit any complications related to the implementation of the protocol.There is limited evidence for the benefit of an ERAS protocol in neurosurgery patients. We show in this study that implementing a multidisciplinary ERAS protocol suited for craniotomy patients is feasible and may reduce the proportion of patients requiring a longer ICU stay significantly. We also demonstrate possible associations with specific protocol components responsible for this improvement in the outcome- decreased pain, less requirement of opioids after surgery, better glycemic control, and earlier mobilization.When our study was initiated in early 2017, there was no evidence that ERAS protocols could be modified and used for neurosurgery patients. A set of suggested guidelines based on non-neurosurgical evidence was presented by Hagan et al., which, although intuitively attractive, would need evidence to be applied to craniotomy patients. ERAS guidelines for spinal surgeries and related studies showing benefits followed , 14. WanERAS protocols are flexible and adapted to individual centers, keeping the basic tenets in place. This is well demonstrated when the two protocols  are compared. Many components were based on previous recommendations and were similar between our groups .The differences are as follows-.Preoperative phase-In our center, relatives are allowed a significant role in patient mobilization, feeding, and patient care - ignorance, fear, and hesitation, delays ambulation, nutritional intake, and rehabilitation during and after the hospital stay. Our preoperative counseling involved an in-depth education of the relatives, including empowering them to question delays in feeding or ambulation orders and removal of foley’s catheter, if any. We educated them on the harmful effects of smoking and alcohol, but abstinence was not essential. As our surgeries were restricted to supratentorial tumors, no added focus than the conventional existed for PONV risk stratification.We administered maltodextrin  drink in different dosages, volumes and times - 200 ml (100 mg) the night before and 100 ml (50 ml) just 2 h. before surgery vs. Wang’s 400 ml maltodextrose-fructose solution on the morning of the day of surgery.We had a more aggressive analgesia plan in Gr E in the form of flupiritine perioperatively  and bilateral complete scalp blocks along with skin site infiltration . This enabled a stricter postoperative VAS guided pain management plan- paracetamol, tramadol, or NSAIDS up to a VAS of 4 and Inj Fentanyl beyond VAS 4.Unlike Wang et al., we did not emphasize on a difference in the ways of stitching/ craniotomy closure or type of suture used between the two groups. Protocols such as antimicrobial preparation, shaving policies, respiratory interventions, goal-directed therapy, and temperature control were the same between Wang et al. and us.We found a significant reduction in the proportion of patients requiring an ICU stay of > 48 h, although there was no significant difference in the total duration of hospital stay. The factors affecting the length of postoperative hospital stay are known to vary widely . Ours isThe ERAS group patients had significantly lower insulin requirements than the controls over 48 h to achieve similar blood glucose targets. Sarin et al. nicely sum the practice points for instituting a protocol for preoperative carbohydrate loading benefits in 2017 . WithoutScalp blocks provide better attenuation of inflammatory and hemodynamic response to craniotomy and better postoperative analgesia than incision site infiltration with local anesthetics . This, aThe strengths of our study are the inexpensive interventions and the pragmatic reproducible endpoints. ‘Availability’ of family members to care for their patients is common in low, middle-income countries (LMIC) where cultural values and norms encourage family involvement . Many syThe lack of randomization is a definite limitation of our study. In 2017, ERAS protocols were allowed to be randomized in gastrointestinal oncosurgeries by our IEC ; evidencNotwithstanding clear protocols for pain assessment, analgesia administration, blood sugar control, extubation, and discharge from ICU, members of the surgical, anesthesia, and ICU teams had been educated of the purported ‘benefits’ of ERAS, and bias is possible. All attempts were made to blind the data collector and analyzer, however. The generalizability of our primary results may depend on institute protocols for ICU discharge. We believe that they will be replicable in centers with limited ICU resources  in other parts of the world. The secondary outcomes, based on internationally accepted protocols, should have external validity.In this study, we have adapted the ERAS protocol to a neurosurgical setting, showing its feasibility and benefits in early discharge from the ICU/ HDU and better pain and blood sugar control postoperatively. However, there was no difference in length of stay in the hospital. ERAS protocols are bundles of care and include various interventions, and further studies are needed to standardize these for the patients undergoing craniotomy such that they may be kept pragmatic and easy to implement.