Garcinia macrophylla Mart. plant extract fractions. An isolate from a fraction of ethyl acetate extract was characterized with spectroscopic data. A new biflavonoid compound was found to have a skeleton of 5,7,4′,5″,7″,3‴,4‴-heptahydroxyflavanone[3-6″] flavones which was named macrophylloflavone (1). The compound was evaluated for its antibacterial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 with cephazolin as a positive control, antioxidant assay against 2,2 diphenyl-1-picrylhydrazyl (DPPH) with ascorbic acid as the positive control, and anti-type 2 diabetes mellitus treatment with metformin as a positive control. The biflavonoid compound exhibited a good inhibition for bacteria and free radical DPPH. Furthermore, biflavonoid compound treatment on the diabetic rats suggested its ability to decrease the blood glucose level. This study provided evidence that the plant has antibacterial, antioxidant, and antidiabetic properties.Investigations of antibacterial, antioxidant, and anti-type 2 diabetes mellitus activities have been carried out on  Degenerative diseases contribute the most number of world's mortalities and morbidities. According to many studies, the main factor causing the degenerative diseases is free radicals, which are actively produced through human body metabolic process –4. DiabeAs an alternative, this study proposed the use of ethnomedicinal plants which have antibacterial, antioxidant, and antidiabetic activities , 12. PlaGarcinia is a plant genus that is known to produce flavonoids in major quantities  270 (C15H11O5) and 284 (C15H8O6).The FTIR spectrum of compound 1H-NMR spectrum of 1 suggested the presence of 13 protons: ten aromatic, two aliphatic, and one olefinic. The aromatic protons appeared at the δH (ppm) 5.96 , 5.98 , 7.07 , 6.64 , 6.43 , 7.10 , 6.25 , 7.33 , 6.91 , and 7.29 . Meanwhile, the aliphatic protons were characterized at the δH (ppm) 5.75  and 4.83 , and the olefinic proton was detected at δH (ppm) 6.39 .The 13C-NMR spectrum of 1 displayed the 30 carbon atoms with the characteristics of two carbonyl carbons, 15 quaternary carbons, and 13 tertiary carbons. The carbonyl carbon signals were detected at the δC (ppm) 197.91 and 183.83. The signals of quaternary carbon appeared at the δC (ppm) 164.81, 165.73, 168.19, 103.19, 130.49, 158.81, 165.73, 157.37, 102.02, 163.31, 162.53, 104.95, 123.34, 146.79, and 150.94. The tertiary carbons were confirmed at the δC (ppm) 82.72, 50.84, 97.43, 96.42, 129.24, 115.61, 115.54, 129.24, 103.37, 99.83, 114.17, 116.84, and 120.55. Indeed, the DEPT data also confirmed the presence of tertiary and quaternary carbons.The δH 5.75 ppm, 1H, d, J = 11.90 Hz) to C-4 (δC 197.91 ppm), C-1′ (δC 130.49 ppm), C-2′ (δC 129.24 ppm), C-6′ (δC 129.24 ppm); H-3  to C-2 (δC 82.72 ppm), C-4 (δC 197.91 ppm), C-1′(δC 130.49 ppm), C-5″(δC 157.37 ppm), C-6″ (δC 102.02 ppm), C-7″ (δC 163.31 ppm). The olefinic H-3″ proton  correlated to C-2″ (δC 165.73 ppm), C-4″ (δC 183.83 ppm), C-10″ (δC 104.95 ppm), C-1‴ (δC 123.34 ppm). The correspondence of ten aromatic protons is displayed in H-6  to C-5 (δC 164.81 ppm), C-8 (δC 96.42 ppm), C-10 (δC 103.19 ppm); H-8  to C-6 (δC 97.43 ppm), C-7 (δC 165.73 ppm), C-9 (δC 168.19 ppm), C-10 (δC 103.19 ppm); H-2′  to C-2 (δC 82.72 ppm), C-4′ (δC 158.81 ppm), C-6′ (δC 129.24 ppm); H-3′  to C-2′ (δC 129.24 ppm); H-5′  to C-3′ (δC 115.61 ppm); H-6′  to C-2 (δC 82.72 ppm), C-2′ (δC 129.24 ppm), C-4′ (δC 158.81 ppm); H-8″  to C-6″ (δC 102.02 ppm), C-7″ (δC 163.31 ppm), C-9″ (δC 162.53 ppm), C-10″ (δC 104.95 ppm); H-2‴  to C-2″ (δC 165.73 ppm), C-3‴ (δC 146.79 ppm), C-4‴ (δC 150.94 ppm), C-6‴ (δC 120.55 ppm); H-5‴ to C-1‴ (δC 123.34 ppm), C-3‴ (δC 146.79 ppm), C-4‴ (δC 150.94 ppm); and H-6‴  to C-2″ (δC 165.73 ppm), C-2‴ (δC 114.17 ppm), C-4‴ (δC 150.94 ppm). The correlations between neighboring protons were indicated in the COSY spectrum data of H-2 with H-3; H-2′ with H-3′; H-5′ with H-6′; and 5‴ with 6‴. The two-dimensional correlation of the HMBC and the COSY cross-peaks is displayed in The HMBC cross-peak between protons and carbons demonstrated two aliphatic protons correlation to several carbons, i.e., H-2  5.75 , 4.83 , 5.96 , 5.98 , 7.07 , 6.64 , 6.43 , and 7.10  exposed the same pattern as the naringenin compound  flavone skeleton pattern. It was clearly confirmed that the δC-6″ appeared in the downfield region and the δC-8″ existed in the upfield region. Referring to the δC-6″ and δC-8″ values of compounds 2, 3, and 4, the δC-6″ value always appeared in the upfield area compared to the δC-8″ value which exists in the downfield area where the linkage occurs in the flavanone[3-8″] flavone skeleton pattern  flavone system.The signals at  pattern –42, 44. 1 was found to have strong antimicrobial activity against both the E. coli and the S. aureus bacteria. The inhibition zones in various concentrations were found to be around 16.65 ± 0.43 to 20.29 ± 0.28 mm (mean ± SD) for E. coli . Meanwhile, for S. aureus, the inhibition zone was around 15.54 ± 0.39 to 23.16 ± 0.32 mm (mean ± SD). The antibacterial properties of flavonoids were based on their ability to inhibit nucleic acid synthesis, cytoplasmic membrane function, energy metabolism, and porins in cell membranes [The classification of antimicrobial strength such as weak, moderate, and strong is indicated by the inhibition zones of <12, 12-20, and >20 mm, respectively . Based oembranes .1 against DPPH free radicals gave the IC50 values of 3.69 ± 0.29 μg/mL (mean ± SD). The strength of an antioxidant's activity is based on the IC50 value criteria; i.e., the IC50 value <10 μg/mL indicates very strong activity, the value 10–50 μg/mL suggests strong activity, the value 50–100 μg/mL implies moderate activity, the value 100–250 μg/mL reveals weak activity, and the value >250 μg/mL signifies no activity [50 value of compound 1 was found to be <10 μg/mL indicating a very strong antioxidant activity. Almost all flavonoid compounds are antioxidants. It is ascribed to their possession of hydroxyl groups in the flavonoids [1 acted as an antioxidant agent donating its hydrogen atoms from the substituent content of the hydroxyl groups towards DPPH free radical, allowing the reduction of the DPPH radical. The reduction of DPPH radicals can be observed through the color change, from purple (DPPH) to yellow  [The antioxidant performance of the compound activity . In thisavonoids . The com1 has the ability to reduce the blood glucose levels in rats with diabetes  = 146.2, p < 0.0001. The main effect between treatment (doses of compound 1) and control (negative and positive controls) referred as F  = 2985, p < 0.0001, produces a significantly different value in blood glucose levels at the posttreatment (mean ± SD), namely, negative control (412.20 ± 7.76), positive control (98.00 ± 1.67), dose 6 μg/kg body weight (171.00 ± 3.81), dose 7 μg/kg body weight (138.00 ± 1.87), and a dose of 8 μg/kg body weight (108.40 ± 3.21). According to the American Diabetes Association, blood glucose levels >180 mg/dL indicate hyperglycemia, while the levels <100 mg/dL indicate hypoglycemia [F  = 239, p < 0.0001 generating the significant force. The analysis of Tukey's post hoc test showed that the positive control has a significant different effect of compound 1 on the dose of 6 μg/kg body weight (p < 0.0001), 7 μg/kg body weight (p=0.0049), and 8 μg/kg body weight (p=0.0055). This means that all variations of compound 1 dosage have antidiabetic activity better than the positive control, but a very significant different result in reducing blood glucose in diabetic rats was shown by compound 1 at a dose of 6 μg/kg body weight. In other words, there were variations in the high and low doses of compound 1 have no effect in decreasing the blood glucose of diabetic rats.The results of the group treatment in decreasing the blood glucose levels are presented in glycemia . Therefo1, i.e., naringenin and luteolin. Flavonoid compounds have double bonds, carbonyl groups, and hydroxy groups which are very important in the antidiabetic activity [α-glucosidase activity, reducing oxidative stress, increasing antioxidant enzymes, and stimulating insulin secretion [1 conclusively proved that the macrophylloflavone (1) has anti-type 2 diabetes property.Decreasing of blood glucose levels in diabetic rats was caused by the activity of flavonoid dimers in compound activity . Naringeecretion , 55. Theβ-cells, and liver hepatocytes cell required the normal rat cells (given code A), as a benchmark in the observations. The data in β-cells, and liver hepatocytes cells. These were ascribed to the presence of necrosis in cells by glucose monohydrate . Alloxan can cause significant damage to these cells by triggering the formation of free radicals that lead to diabetes in rats [The histopathological data of kidney tubular cells, pancreatic  in rats .1  reduced the necrosis in kidney tubular cells (1 could repair the tubular cell damage in the kidney. The number of necrotic cells in the kidney tubules in the treatment group with 8 μg/kg body weight dose (B4) revealed the similarities with the normal rat kidney tubules (A) (p < 0.05). The kidneys contribute to glucose homeostasis through the processes of gluconeogenesis, glucose filtration, glucose reabsorption, and glucose consumption [1 have produced a positive effect in preventing damage to kidney tubular cells so that the appearance of tubular cells is closer to the appearance of tubules under normal conditions  (1 with a dose of 8 μg/kg body weight (B4) has to afford no difference in the number of normal pancreatic β-cells (A) (p < 0.05). Application of the compound 1 was observed to increase the formation of β-cells in the pancreas  . Adminispancreas . This wapancreas .1 with various doses  (1 with dose of 7 μg/kg body weight (B3) and dose of 8 μg/kg body weight (B4) resulted in a number of liver hepatocyte necrosis cells same as the number of normal hepatocyte necrosis cells (A) (p < 0.05). The compound 1 was shown not to damage liver hepatocyte cells (not toxic) and regenerate liver hepatocyte cells in the normal range (The liver hepatocyte cell damage decreased after giving the compound  and B4) . The comal range . Antioxi1) as new biflavonoid compound with linkage 3-flavanone-6″-flavone has been successfully isolated from the ethyl acetate fraction from the stem bark of G. macrophylla plant. The in vitro evaluation of 1 suggested strong antibacterial and also antioxidant activities. More importantly, the in vivo assay exhibited the ability of 1 to decrease blood glucose levels in the diabetic rats to the normal level.The macrophylloflavone (


SI) gene, driven by stem-specific promoters (A2 or LSG) with a vacuole-targetted signal peptide, was transformed into the sorghum inbred line (T×430).Sugar content is critically important in determining sugar crop productivity. However, improvement in sugar content has been stagnant among sugar crops for decades. Sorghum, especially sweet sorghum with high biomass, shown great potential for biofuel, has lower sugar content than sugarcane. To enhance sugar content, the sucrose isomerase  were crossed with sweet sorghum . Total sugar contents (over 750 mM), were notably higher in F1, and F2 progenies than the control Rio (480 mM). The sugar contents of the engineered lines (over 750 mM), including T0, T1, F1, and F2, are surprisingly higher than that of the field-grown sugarcane . Additionally, analysis of physiological characterization demonstrated that the superior progenies had notably higher rates of photosynthesis, sucrose transportation, and sink strength than the controls.The study demonstrated that transgenic lines of grain sorghum, containing 50–60% isomaltulose, accumulated up to eightfold (1000 mM) more total sugar than the control T×430 did (118 mM) in stalks of T0, and T1) and hybrid sorghum , demonstrating that sorghum can accumulate as high or higher sugar content than sugarcane. This research illustrates that the SI gene has enormous potential on improvement of sugar content in sorghum, particularly in hybirds and sweet sorghum. The substantial increase on sugar content would lead to significant financial benefits for industrial utilization. This study could have a substantial impact on renewable bioenergy. More importantly, our results demonstrated that the phenotype of high sugar content is inheritable and shed light on improvement for other sugar crops.The genetic engineering approach has dramatically enhanced total sugar content in grain sorghum (TThe online version contains supplementary material available at 10.1186/s13068-021-01907-z. Sugar yield, largely impacted by biomass and sugar content, is a key determinant of economic sustainability for sugar crops. In recent decades, improvement on sugar yield has been achieved almost entirely by increasing biomass –3, despi4, drought tolerant, high biomass, and high water use efficiency crop that produces stalks up to five meters tall, accumulating sucrose . However, current sweet sorghum varieties, producing comparatively low sugar content (around 500 mmol/L), urgently require plant breeders to improve sugar accumulation in stalks for biofuel  containing 0.2 µCi (14C) sucrose and unlabelled sucrose to the desired concentration. At each time point, vesicles from one reaction mixture were collected on 0.45-µm filters and rinsed three times with 0.6 mL of assay buffer containing only unlabelled sucrose (1 mM). The accumulated radioactivity was measured by scintillation spectrometry. The difference between samples with and without 5 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was defined as ∆pH-dependent sucrose transport.Transport assays were conducted at 12 °C using three replicate reactions per treatment . Briefly, for each reaction mixture, 20 µL of resuspended PMVs were diluted into 400 µL of assay buffer . The homogenate was immediately centrifuged at 10 000 g for 15 min at 4 °C. The supernatant was immediately desalted on a PD-10 column  that was pre-equilibrated and eluted using the extraction buffer. Protein concentration was assayed by the Bradford reaction using a Bio-Rad kit  with bovine serum albumin standards. SI activity was measured by incubating enzyme extract with 292 mM sucrose solution in 0.1 M citrate–phosphate buffer (pH 6.0) at 30 °C.The SI enzyme was extracted by grinding the frozen cells in a chilled mortar using three volumes of extraction buffer that contained 0.1 M Hepes–KOH buffer(pH7.5), 10 mM MgClAdditional file 1: Fig. S1. Transgenic sorghum lines were grown in a PC2 glasshouse. (a) 1 week; (b) 2 weeks; (c) 3 weeks; (d) 5 weeks; (e) 7 weeks; (f) 10 weeks in the glasshouse; (g) mature T×430; (h) mature A5; (f) mature L9. Fig. S2. The Construct Used for Gene Transformation. Stem-specific Promoter: either A1 resulting in high gene expression in the mature stalk or LSG2 resulting in high gene expression in the loading sucrose section of the stalk. Vacuole leading: encoding a propeptide to guide SI gene products to vacuole, where sucrose accumulates. Non-silence target gene: sucrose isomerase without motifs gene silencing in plants. Multiple terminators: three recombined terminators complex to guarantee the proper termination of gene transcription. Fig. S3. PCR screening of sucrose isomerase gene in transgenic LSG2 lines. The agarose gel displayed PCR results of transgenic lines. M: DNA ladder; NC: negative control; Transgenic lines L1, L2, L3, L4, L5, L7, L9, L14, and L16 were positive of the sucrose isomerase gene; Transgenic lines L6, L8, L10, L11, L12, L13,L15, and L17 were negative of the sucrose isomerase gene. Fig. S4. Sugar profile of T1 L9 lines. Sugars, including isomaltulose, were measured 20-day post-anthesis in the middle section of internode 4 (counted from top). L9 is one of the top lines in T0 transgenic lines. L9-2, 9-3, 9-6, 9-7, 9-11, 9-12 are isomaltulose positive samples. Nil-L9-4, 9-8 and 9-9 are null-segregant samples in T1 generation. T×430-1, T×430-2, and T×430-3 are the non-transformed control. Fig. S5. PCR screening of sucrose isomerase gene in T1 lines. The agarose gel displayed PCR results of transgenic lines and controls. M: 1 Kb DNA ladder; Positive A5 T1 progenies: A5-2 and A5-4; Positive L2 T1 progenies: L2-1, L2-3 and L2-6; Positive L9 T1 progenies: L9-2, and L9-4; T×430-1, T×430-2 are non-transgenic control samples; PC: positive plasmid control (LSG2). Table S1. Sugar profile of controls and positive transgenic lines with isomaltulose. Table S2. Sugar profile of F1 hybrid lines of R9188 X L9.


Stomolophus sp. 2 are not well-known. The digestion study was critical to explain its ecology and bloom success. Different enzymes are involved in food digestion, which hydrolyze carbohydrates, proteins, and lipids. This study detected lipolytic activity in enzymatic extracts from gastric pouches of Stomolophus sp. 2 collected in the summer of 2013 at Bahía de Kino, Sonora, México (28°47′47″N 111°57′25″W). Lipase/esterase activity showed optimal pH at 11.0 and 50–60 °C with a half-life (t1/2) of 33 min at 55 °C, whereas halotolerance of this activity was recorded from 0-4 M NaCl. Metal ions Ca2+ and Mn2+ did not affect the activity, but Mg2+ decreased it 14.2% ± 3.15, while chelating agents as ethylenediaminetetraacetic acid reduced the activity 8.55% ± 2.13. Inhibition of lipase/esterase activity with tetrahydrolipstatin and paraoxon-ethyl decreased the activity 18.2% ± 2.3, and 62.80% ± 0.74, respectively, whereas phenylmethanesulfonyl fluoride (a protease inhibitor) did not affect it. The enzyme displayed a higher specificity for short-chain triglycerides, but triolein, coconut oil, olive oil, and fish oil were hydrolyzed. For the first time, phospholipase activity from the gastric pouch of Stomolophus sp. 2 was detected using L-α-phosphatidylethanolamine from chicken egg yolk as a substrate. These results suggest that Stomolophus sp. 2 hydrolyze several kinds of lipids, and lipolytic enzymes are active at alkaline pH under different saline conditions, which may be essential to digest different preys.The digestive system and metabolism of the cannonball jellyfish  Stomolophu meleagris belongs to cnidarians of the phylum Coelenterata, class Scyphomedusae, order Rhizostomeae, family Stomolophidae, and genus Stomolophus  although no amylase, lactase or cellulose were found (Mastigias papua)  and alkaline and acid phosphatase, chymotrypsin and lipase activity in cannonball jellyfish (S. meleagris) (S. meleagris gastric cirri.Previous studies have investigated re found  while ens papua) . Anotherleagris) . BodanskDue to the kind of prey that jellyfish eats, and the little knowledge about its digestion, biochemical identification of lipolytic enzymes (EC 3.1.1.X) is important to understand the nutrition and life cycle of jellyfish species. Lipases and esterases are key hydrolases in animal digestion.sn-phospholipid to release fatty acids and lysophospholipids  . Esterasholipids .Stomolophus sp. 2, using synthetic and natural oils  to know the capacity of lipolytic enzymes in the digestive system from jellyfish; subsequently, if in whole-body jellyfish polyp, ephyra and medusa stages the lipase activity was observed; then, if lipolytic activity found in gastric pouches from the adult phase of Stomolophus sp, 2 corresponded to digestive lipase/esterase enzymes.Previous studies on cannonball jellyfish  have reported lipolytic activity with synthetic substrates , but no Stomolophus sp. 2.Therefore, this study aimed to contribute to a better understanding of cannonball jellyfish lipid digestion, showing the characterization of lipolytic activities from the digestive system of the blue cannonball jellyfish p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4), p-nitrophenyl valerate (C5), p-nitrophenyl octanoate (C8), p-nitrophenyl decanoate (C10), p-nitrophenyl laurate (C12), p-nitrophenyl myristate (C14), p-nitrophenyl palmitate (C16), p-nitrophenyl stearate (C18), vinyl butyrate (V4), vinyl laurate (V12), tripropionin (TC3), tributyrin (TC4), trioctanoin (TC8), triolein (TC18), methylumbelliferyl-butyrate (MUF-B), L-α-phosphatidylcholine from egg yolk and fish oil; Inhibitors and detergent: tetrahydrolipstatin (THL), paraoxon-ethyl (E600), phenylmethanesulfonyl fluoride (PMSF), N-lauroylsarcosine sodium salt (NLS) and sodium dodecyl sulfate (SDS); buffers: 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS). Substrates, inhibitors and buffers were purchased from Sigma-Aldrich-Fluka . Coconut oil and other reagents were obtained from local suppliers.Substrates used: Stomolophus sp. 2 were collected in the spring of 2013 from surface waters at Bahía de Kino, Sonora, México (28°47′47″N 111°57′25″W)  , using a57′25″W) ; then, tStomolophus sp. 2, which were homogenized at 4 °C to low stirring speed in one mL of distilled water for 30 min; then, samples were centrifuged 10,000 g at 4 °C for 15 min to eliminate tissue debris. The supernatant was stored at −20 °C for further analyses.The enzymatic extract was prepared from 50 mg of freeze-dried gastric pouches from p-nitrophenyl decanoate (C10) as substrate. The release of p-nitrophenol was monitored at 415 nm during enzymatic hydrolysis. The reaction mixture was prepared by diluting 1:10 (v/v) a solution of 10 mM of C10 (dissolved in 2-methyl-2-butanol) with a buffer solution ; 40 µL (0.88 mg/mL) of the jellyfish gastric pouch extract with 160 µL of the reaction mixture were placed in a microplate. The release of p-nitrophenol was monitored at 415 nm at 37 °C for 15 min and at time intervals of 30 s. The reaction rate was calculated with the absorbance curve slope versus time by using a molar extinction coefficient of 22,931 cm−1 M−1 with 17 mM N-lauroylsarcosine sodium salt  esters was determined at 37 °C in 50 mM Tris-HCl buffer, pH 8.0. Fatty acid chain lengths from C2 to C18 were used as substrates (10 mM) dissolved in 2-methyl-2-butanol was diluted 1:10 (v/v) with buffer. When the synthetic substrate was C2, C4, and C5 the buffer solution was prepared without N-lauroylsarcosine sodium salt detergent—extinction molar coefficients were 22,233 cm−1 M−1 and 22,931 cm−1 M−1 for solutions with and without detergent, respectively. One unit of activity was defined as the amount of enzyme that releases 1 µmol of fatty acid per minute in the assay conditions.The acyl chain length specificity towards different The fatty acyl chain length specificity was measured by using two different vinyl esters solutions: 73.6 mM vinyl butyrate (V4), and 35.9 mM vinyl laurate V12), both were dissolved in 2.5 mM Tris-HCl, pH 8.0 buffer. The activity was determined by titration using pH-Stat method at constant pH value (8.0) at 37 °C. The reaction was done in a glass vessel containing 82 mg of lyophilized jellyfish gastric pouch content dissolved in 10 mL of substrate. The free fatty acids released were titrated with 0.1 M NaOH . The act2. When oils and triolein were used, the substrates were pre-emulsified with gum arabic (3% w/v) . Each assay was performed at 37 °C containing 2.5 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM CaCl(3% w/v) . The actPhospholipase activity was tested by the release of fatty acids and measured by pH-Stat  titration method under mechanical stirrer in a 10 mL reaction vessel at 37 °C, adding 0.1 M NaOH, using L-α-phosphatidylcholine from chicken egg yolk as substrate . Hydrolyn = 20) and a mixture of them were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), containing 12% acrylamide under non-reducing conditions  . After pp-nitrophenyl decanoate. To evaluate the ionic strength effect on lipase activity, NaCl concentrations were adjusted (0–4 M) in the buffer solution (50 mM Tris–HCl pH 8.0) at 37 °C. To identify optimum pH, different buffers were used: 50 mM MES, pH 6.0; 50 mM MOPS, pH 7.0; 50 mM Tris-HCl, pH 8.0; 50 mM CHES, pH 9.0; 50 mM sodium carbonate-sodium bicarbonate, pH 10; 50 mM CAPS, pH 11.0 and 50 mM KCl-NaOH pH 12.0 at 37 °C. Different extinction molar coefficients were used to calculate the lipase/esterase activity, ε . For optimum temperature, lipase activity was evaluated in the range from 25–80 °C for 15 min reaction at pH 8.0. To measure lipolytic enzyme thermostability, jellyfish gastric pouch extract was incubated at 25–60 °C for 60 min. The residual activity was recorded by the spectrophotometric assay as previously described.Operational variables from the cannonball jellyfish lipases were measured using 2+, Mn2+, Mg2+ andthe metal-chelating agent ethylenediaminetetraacetic acid (EDTA) was evaluated on lipase/esterase activity in 50 mM Tris-HCl buffer (pH 8.0) using p-nitrophenyl decanoate (C10) as substrate and 40 µl (0.88 mg/mL) of jellyfish gastric pouch extract. The inhibitory effect was tested with tetrahydrolipstatin (THL), paraoxon-ethyl (E600), phenylmethanesulfonyl fluoride (PMSF) on dimethyl sulfoxide. The jellyfish gastric pouch extract was incubated at 37 °C for 60 min with 1 mM of each metal ions and inhibitor solutions; after that, the lipase/esterase activity was measured.The effect of various metal ions: Cap < 0.05.The results are represented as mean of three biological samples. One-way non-parametric ANOVA was used to evaluate lipase/esterase and phospholipase activity in all assays under different reaction conditions. The difference of means between pairs was resolved using confidence intervals of Fisher’s least significant difference (LSD) test with the statistical software program (SigmaPlot v. 10.0). Differences were reported as statistically significant when n = 20); an average of extract lipase activity from the same organisms was 0.80 ± 0.30 U/mg freeze-dried gastric pouch for p-nitrophenyl butyrate; and 0.23 ± 0.10 U/mg freeze-dried gastric pouch for p-nitrophenyl decanoate. Proteins and profiles of lipases/esterases were similar among individuals; both profiles are shown in The average protein concentration of jellyfish gastric pouch extract was 0.26 ± 0.10 of protein/mg freeze-dried gastric pouch (p-nitrophenyl decanoate.Lipolytic activity was tested in a wide range of pH (6.0–12.0), since pH 7.0 lipolytic activity had risen until it reached optimum activity at pH 11.0, and then the activity decreased . At pH 61∕2) at 55 °C at 33 min , as salt concentration increased, lipase activity also increased; a maximum value (94.16 ± 0.56 mU/mL) was observed at 4 M NaCl (i = 4) with respect to 53.5 ± 0.31 mU/mg at i = 0 (NaCl absence) ; the activity was influenced by ionic strength (absence) .Lipolytic activity was not significantly affected by ions except for magnesium ions that reduced lipolytic activity, 15.00 ± 3.15% . Howeverp-nitrophenyl (p-NP) esters as substrates with different acyl chain lengths. Lipase activity for short-chain acyl substrates as p-NP acetate (C2), p-NP butyrate (C4), and p-NP valerate, (C5) 0.26 ± 0.01., 0.84 ± 0.02, and 0.45 ± 0.03 U/mg of protein were shown, respectively. No statistically significant results were found among medium acyl chain substrates: p-nitrophenyl octanoate (0.27 ± 0.02), and p-nitrophenyl decanoate (0.23 ± 0.01). Meanwhile, lipolytic activity on large chain acyl substrates, as p-NP laurate (C12), p-NP myristate (C14), p-NP palmitate (C16), and p-nitrophenyl stearate (C18) were 0.24 ± 0.04, 0.13 ± 0.01, 0.08 ± 0.01 and 0.18 ± 0.06 U/mg of protein, respectively  as substrate and reported as lipase  (Todarodes pacificus), flying squid (Ommastrephes bartramii) and marine sponge  which showed an optimal activity from 35–40, 25 and 40 °C, respectively. The thermostability results indicated that lipolytic activity reduced rapidly at 60 °C although at 25 °C it was maintained at 100% for one hour whereas at 50 °C, 33.8% of the activity was lost   but not was lost . This daStaphylococcus sp. lipase  and in absence of salt, halotolerant enzymes remain active over a broad range of NaCl concentration without any specific salt dependence . Negativ. lipase . The coa. lipase . Jellyfi+2 because 100% of the activity was maintained without Ca+2, and the activity for 1 mM CaCl2 was 98.5% ± 2.5. In vertebrates, cationic cofactors are required for lipase activation; however, it was not observed in most invertebrates, including the white leg shrimp (Litopenaeus vannamei) (Todarodes pacificus)  was reported with Mg2+   and Pacicificus) . Nonetheith Mg2+ . In thisMg2+  have been recognized as serine lipase inhibitors since they react with the catalytic serine residue; E600 inhibited 62.80 ± 0.74% and THL only 18.57 ± 2.35% . PMSF dir miehei . In Baci lipases . Lipase Stomolophus sp. 2) gastric pouches were able to hydrolyze short-chain triacylglycerides better than long ones, in contrast to other marine invertebrates as the whiteleg shrimp (Litopenaeus vannamei) digestive lipase (PVL) with 475.8 ± 4.9 U/mg protein for tributyrin and 1,787 ± 7.9 U/mg protein for triolein (Carcinus mediterraneus), the activity was four times higher on TC4 compared to that of olive oil (Hexaplex trunculus) with 400 U/mg protein for tributyrin and 100 U/mg protein for olive oil  (30.0 ± 0.3 U/g fresh tissue) and octopus (Octopus marginatus) (25.0 ± 1.25 U/g fresh tissue) (Lipolytic digestive enzymes from jellyfish (triolein . In the live oil . The tenlive oil . In addi tissue) .Aphis melifera) (Mesobuthus tamulus and Pandinus imperator) (Phospholipase activity has been reported in different tissues of Scyphozoa (umbrella and tentacles). In cnidarian specimens, class Anthozoa, Hydrozoa, Scyphozoa, and Cubozoa, different levels of phospholipase activity have been found in tentacles and acontia, which are known by the high amount of nematocysts and whole-body extract organisms , as wellelifera) , scorpioperator) , and cniperator) .Stomolopohus sp. 2. Chicken egg yolk is a rich source of phospholipids with an average of neutral lipids (65%), phospholipids (31%), and cholesterol (4%)  . Padillarol (4%) . Thus, irol (4%)  and preyFish eggs and larvae and cypris larvae of barnacle are considered as jellyfish prey; their total lipid composition are neutral lipids and phospholipids, which represent 8.1–10.4% and 4.4–5.3% of total dry weight from the organisms mentioned . LikewisOur results revealed that the cannonball jellyfish digestive system contained at least three lipolytic enzymes that displayed activity against several short and long fatty acids, of which the short ones are their preferential substrate. Moreover, phospholipase activity was observed in the gastric pouch, which had not been reported before, as well as halotolerant characteristics of lipolytic enzymes (lipases and esterases). This study is a pioneer to understand the digestive capacity of jellyfish species to digest several lipid substrates.10.7717/peerj.9794/supp-1Supplemental Information 1p-nitrophenol esters, (6) Lipase/esterase activity of jellyfish gastric pouch extract on triacylglycerols, phospholipids and vinyl esters, (7) Effect of different ions and inhibitors on lipase/esterase activity of jellyfish gastric pouch extract.(1) Effect of pH on lipase/esterase activity, (2) Effect of temperature on lipase/esterase activity of jellyfish gastric pouch extract, (3) Thermostability of lipase/esterase activity of jellyfish gastric pouch extract, (4) Effect of NaCl on lipase/esterase activity of jellyfish gastric pouch extract, (5) Fatty acid chain length specificity of jellyfish gastric pouch extract using Click here for additional data file.10.7717/peerj.9794/supp-2Supplemental Information 2Stomolophus sp 2 gastric pouch extract; RL: Rhizomucor miehei lipase; T: inhibition with 1 mM tetrahydrolipstatin, E: inhibition with 1 mM paraoxon-ethyl (E600) and P: inhibition with 1 mM phenylmethanesulfonyl fluoride (PMSF). Enzymes were incubated at 37 °C for 1 h; then 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 7.5 mA.Zymograms were developed using methylumbelliferyl-butyrate (MUF-B) as substrate. LE: lipolytic enzymes from Jellyfish Click here for additional data file.10.7717/peerj.9794/supp-3Supplemental Information 3MM, Molecular Mass. 1–6= organism ID.Click here for additional data file.10.7717/peerj.9794/supp-4Supplemental Information 4MM, Molecular Mass. 7–13, organism ID. 14, jellyfish gastric pouch extract pooled from 20 organisms.Click here for additional data file.10.7717/peerj.9794/supp-5Supplemental Information 57–13= organism ID. 14, jellyfish gastric pouch extract pooled from 20 organisms.Click here for additional data file.10.7717/peerj.9794/supp-6Supplemental Information 6Imagen shows reagents, and pH/time conditions used during lipolysis.Click here for additional data file.


We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4  •CDK8 regulates IL-6-mediated STAT3 S727 phosphorylation in primary human T cells•CDK8 controls STAT3 activity by limiting its resident time at gene loci•CDK8 inhibition increases IL-6-mediated Th17 differentiation How IL-6 elicits its immune pleiotropic activities is not fully understood. Martinez-Fabregas et al. show that CDK8 represses IL-6-mediated transcription by limiting STAT3 resident time at the gene loci. By regulating CDK8 expression levels, immune cells can adapt their responses to STAT3-activating cytokines. Cytokines are critical orchestrators of innate and adaptive immunity . DespiteSTATs can be modified in conserved Tyr or serine (Ser) residues . AlthougListeria monocytogenes infection, highlighting the relevance of STAT1 Ser phosphorylation in modulating IFNγ response in macrophages (Interleukin-6 (IL-6) represents a classical paradigm for cytokine functional pleiotropy. IL-6 acts as a central regulator of the immune response by triggering both pro-inflammatory and anti-inflammatory responses . IL-6 drrophages . Howeverin vitro. Overall, our studies identify a STAT3 regulatory mechanism in T cells, whereby CDK8 and CDK9 modulate STAT3 processivity by controlling its chromatin binding dwell time and transcriptional activity. These observations suggest ways to manipulate IL-6- and STAT3-mediated responses by fine-tuning CDK8/9 activities.In this study, we set out to characterize how the signaling initiated by IL-6 in human T cells leads to its functional pleiotropy. We detected both STAT3 Tyr and Ser phosphorylations in response to IL-6 stimulation, with STAT3 Ser phosphorylation exhibiting a delayed activation. Using a battery of small molecule inhibitors, we identified CDK8 and CDK9 as the kinases driving Ser phosphorylation of STAT3 upon IL-6 stimulation in T cells. Using proximity ligation studies, we confirmed the increased interaction between STAT3 and CDK8 and CDK9 in the nucleus upon IL-6 stimulation. Inhibition of the activity of these two kinases resulted in a more robust interaction with STAT3, even in the absence of IL-6 stimulation. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) studies revealed a global increase in STAT3 chromatin binding upon inhibition of CDK8, resulting in the induction of a larger gene expression program by IL-6. In agreement with this enhanced gene expression program, IL-6 induced a more robust differentiation of Th-17 cells upon CDK8 inhibition + and CD8+ T cells stimulated with IL-6. IL-6 receptor expression varies significantly among different T cell populations and environmental contexts . We selected Th-1 cells due to their significance in immunity and ability to expand  studies A.Figure The combined analysis from six independent biological replicates of unstimulated versus HyIL-6-stimulated Th-1 cells identified 17,935 phosphosites on 4,196 proteins . Among tin vitro or in vivo studies.HyIL-6 triggers the Tyr and Ser phosphorylation of STAT1 and STAT3 in T cells. Although JAK1 contributes to the Tyr phosphorylation of STAT1/3, the kinase responsible for STAT1/3 Ser phosphorylation in T cells is currently not known. To identify this kinase, we used a panel of inhibitors targeting signaling pathways previously shown to regulate STAT1 and STAT3 Ser phosphorylation in different cellular systems . Tofacit+ and CD8+ T cells . In untreated cells, we detected very low levels of STAT3/CDK8 , a technique that allows the detection of protein complexes at endogenous levels without the need of protein overexpression or labeling that could interfere with their binding partners . ActivatAT3/CDK8 A and STAAT3/CDK8 B. Upon HAT3/CDK8 A and 4B,AT3/CDK8 B.Figure + T cells were stimulated with HyIL-6 for 30 min in the presence of different CDKs inhibitors. Levels of STAT3/CDK8 and STAT3/CDK9 complexes were measured by PLA analysis. As before, HyIL-6 stimulation led to a significant increase in the number of STAT3/CDK8 and STAT3/CDK9 complexes, when compared to unstimulated cells  modulates its transcriptional activity . Thus, wOur data have highlighted a critical role of CDK8 in regulating STAT3 Ser phosphorylation and nuclear dynamics in human Th-1 cells A, and 4CGSE21670) .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Ignacio Moraga Gonzalez (This study did not generate new unique reagents.www.proteomexchange.com). The raw and processed ChIP-seq and RNA-seq data are deposited to GEO: GSE147399.The phosphoproteomic data have been deposited in the ProteomeXchange: PXD020964  from healthy donors were purified from buffy coats acquired from the Scottish Blood Transfusion Service and used for signaling and phosphoproteomics experiments.In vitro polarized human Th-1 cells generated from human PBMCs acquired from StemCell Technologies (Cat#70025) were used for RNA-seq and ChIP-seq experiments.www.lgcstandards-atcc.org).Hut78 (cat#TIB-161) and HEK293T (cat#CRL-11268) cells were obtained from ATCC (Spodoptera frugiperda (Sf9) cells grown in SF900III media  and used to infect Trichoplusiani ni (High Five) cells grown in InsectXpress media  for protein expression. After 48h infection, secreted protein was captured from High Five supernatants using HisPur Ni-NTA resin  affinity chromatography, concentrated, and purified by size exclusion chromatography on a Enrich SEC 650 1 × 300 column (Biorad), equilibrated in 10 mM HEPES (pH 7.2) containing 150 mM NaCl. Recombinant HyIL-6 was purified to greater than 98% homogeneity.HyIL-6  cloned i+ and CD8+ T cells isolation, 1 × 108 PBMCs per donor were stained with anti-CD4FiTC  or anti-CD8FITC  antibodies and isolated by magnetic activated cell sorting  using anti-FiTC microbeads  according to manufacturer’s instructions to a purity ≥ 99%.Peripheral blood mononuclear cells (PBMCs) from healthy donors were purified from buffy coats (Scottish Blood Transfusion Service) by density gradient centrifugation following manufacturer’s instructions . For CD45 cells/mL. Cell were then stimulated with different concentrations to obtain the dose-response curves. After stimulation cells were fixed with 2% formaldehyde for 10 minutes at RT.For dose-response experiments of STAT1 or STAT3 phosphorylation, 96-well plates were prepared with 30 μL of cells at 5 × 10For kinetics experiments, cell suspensions were stimulated with saturating concentrations of the cytokines (10 nM HyIL-6) as indicated and cells finally fixed with 2% formaldehyde for 10 minutes at RT.BV510 , anti-CD4PE , anti-CD8AF700 , anti-pSTAT1-Tyr701AF647 , anti-pSTAT1-Ser727AF488 , anti-pSTAT3-Tyr705AF488  and anti-pSTAT3-Ser727AF647 . Cells were analyzed in a CytoFlex S flow cytometer (Beckman Coulter) with the individual cell populations being identified by their barcoding pattern and mean fluorescence intensity (MFI) for the different forms of STAT1 or STAT3 measured.After fixation, cells were collected by centrifugation at 1200 rpm for 5 min, formaldehyde blocked by washing the cells with 200 μL of PBS containing 5 mg/ml BSA (PBSA) and collected again by centrifugation at 1200 rpm for 5 min. Then, cells were resuspended and permeabilized in ice-cold methanol for 20 minutes on ice. Cells were then fluorescently barcoded  using a BV510 , anti-CD4PE , anti-CD8AF700 , anti-pSTAT1-Y701AF647 , anti-pSTAT1-S727AF488 , anti-pSTAT3-Y705AF488  and anti-pSTAT3-S727AF647 , anti-pSTAT4-Y693AF488 , anti-pSTAT5-Y694AF647 , anti-pSTAT6-Y641AF488 , anti-pERK-T202/Y204AF488 , anti-pAKT-S473AF488 , anti-pAKT-T308AF647 , anti-pP90RSK-S380AF488 , anti-pS6R-S240/S244AF488 , anti-pS6R-S235/S236AF647 , anti-pZAP70-Y319/pSYK-Y352AF647 , anti-pCREB-S133AF488 , anti-pHIS3-S10AF647 , anti-pGSK3β-S9AF647 ,anti-pCFOS-S32AF647 , anti-IRF1AF647 , anti-IRF4AF647 , anti-IRF7AF647 , anti-GATA3AF488 , anti-TBETAF647 , anti-HIF1αAF488 , anti-MYCAF488 , anti-O-GlcNACAF647 , anti-STAT3APC  and anti-PLCγ1AF647 . Cells were analyzed in a CytoFlex S flow cytometer (Beckman Coulter) with the individual cell populations being identified by their barcoding pattern and mean fluorescence intensity (MFI) measured.Resting PBMCs isolated as described before from buffy coats or upon activation for three days with ImmunoCult Human CD3/CD28 T Cell Activator  following manufacturer instructions in the presence of 20 ng/mL IL2  were starved for 24 hours in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum  and then stimulated with 10nM HyIL-6 or 0.1 μg/mL anti-CD3 (Biolegend #300438) and 20 ng/mL IL2 (Novartis #709421). Then, cells were fixed with 2% formaldehyde, permeabilized with ice-cold methanol and barcoded as described above. Finally, cells were pooled and stained with anti-CD3+ T cells were labeled with anti-CD4-FiTC antibody  and isolated from human PBMCs by magnetic activated cell sorting  using anti-FiTC microbeads  following manufacturer instructions. Subsequently, resting CD4+ T cells were activated under Th-1 polarizing conditions. Briefly, 3x107 resting human CD4+ T cells per donor were primed for three days with ImmunoCult Human CD3/CD28 T Cell Activator  following manufacturer instructions in the presence of 20 ng/mL IL2 (Novartis #709421), 20 ng/mL IL12  and 10 ng/mL anti-IL4 . Then, cells were split into three different conditions light SILAC media  and 84mg/mL L-Arginine R0 ), medium SILAC media  and 103 mg/mL L-Arginine U-13C6 R6 ) and heavy SILAC media  and 105.8 mg/mL L-Arginine U-13C6,U-15N2 R10 ) prepared in RPMI SILAC media  supplemented with 10% dialyzed FBS , 5 mL L-Glutamine , 5 mL Pen/Strep , 5 mL MEM vitamin solution , 5 mL Selenium-Transferrin-Insulin  and expanded in the presence of 20 ng/mL IL2 and 10 ng/ml anti-IL4 for another 10 days in order to achieve complete labeling. Incorporation of medium and heavy version of Lysine and Arginine was checked by mass spectrometry and samples with an incorporation greater than 95% were used. After expansion, cells were starved without IL2 for 24 hours before stimulation with 10 nM HyIL-6 in the presence or absence of 2 μM MSC2530818  for 15 minutes. Cells were then washed three times in ice-cold PBS, mix in a 1:1:1 ratio, resuspended in SDS-containing lysis buffer (1% SDS in 100mM Triethylammonium Bicarbonate buffer (TEAB)) and incubated on ice for 10 minutes to ensure cell lysis. Then, cell lysates were centrifuged at 20000 g for 10 minutes at +4°C and supernatant was transferred to a clean tube. Protein concentration was determined by using BCA Protein Assay Kit , and 10 mg of protein per experiment were reduced with 10mM dithiothreitol  for 1 hour at 55°C and alkylated with 20mM iodoacetamide  for 30 min at RT. Protein was then precipitated using six volumes of chilled (−20°C) acetone overnight. After precipitation, protein pellet was resuspended in 1mL of 100mM TEAB and digested with Trypsin  and digested overnight at 37°C. Then, samples were cleared by centrifugation at 20000 g for 30 min at +4°C, and peptide concentration was quantified with Quantitative Colorimetric Peptide Assay .Resting CD4Digested samples were fractionated to reduce sample complexity and increase the efficiency of phosphopeptide enrichment. Briefly, peptides (approx. 3.5 mg per sample) were resuspended in 200uL Buffer A (10mM ammonium formate), separated on a XBridge Peptide BEH column  after initially trapped on a XBridge trap cartridge  using an Ultimate 3000 RSLCnano system (Thermo Scientific). Peptides were resolved using a gradient  of Buffer A (10mM ammonium formate) and Buffer B : 8% Buffer B for 6 min, 8%–45% Buffer B for 54 min, 45%–100% Buffer B for 5 min, 100% Buffer B for for 16 min and 100%–2% Buffer B for 21 min. 80 Fractions were collected using a WPS-3000FC autosampler (Thermo Scientific) of 1 minute (0.8 ml) duration from 1-80 min over the chromatogram. These fractions were then concatenated to 20 fractions to provide a similar quantity of peptide per fraction based on the online (U3000 Variable Wavelength Detector) (Thermo Scientific) UV values of the eluted peptides at 220 nm. These concatenated fractions were taken to dryness  prior to suspending for nLC-MS analysis.2, 150 min acquisition) consisting of full scans and mass range (m/z) between 350 to 1600 (m/z) for MS search and 200 to 2000 (m/z) for MS2 search was used. For the MS scan the Q Exactive Plus Mass Spectrometer was operated in a data dependent acquisition mode, resolution of 70,000 with a lock mass set at 445.120024 and max fill time of 20 ms. For the MS2 scan Q Exactive Plus Mass Spectrometer was operated in a centroid mode, resolution of 15,000 with isolation window = 1.4 (m/z), normalized collision energy = 27, max fill time of 100 ms and dynamic exclusion of 45.0 s.Phosphopeptide enrichment in the peptide fractions generated as described above was carried out using MagResyn Ti-IMAC following manufacturer instructions . Phosphopeptide samples were analyzed using a nanoflow liquid chromatography system  coupled to a Q Exactive Plus Mass Spectrometer (Thermo Scientific). Samples (10 μl) were loaded onto a C18 trap column and washed for 5 minutes with 0.1% formic acid. Peptides were resolved using a gradient  of buffer A (0.1% formic acid) and buffer B (80% acetonitrile in 0.08% formic acid): 5% buffer B for 5 min, 5%–35% buffer B for 125 min, 35%–98% buffer B for 2 min, 98% buffer B for 20 min, 98%–2% buffer B for 1 min and 2% buffer B for 17 min. Peptides, initially trapped on an Acclaim PepMap 100 C18 colum , were separated on an Easy-Spray PepMap RSLC C18 column , and finally transferred to a Q Exactive Plus Mass Spectrometer via an Easy-Spray source with temperature set at 50°C and a source voltage of 2.0kV. For the identification of peptides, a top 15 method  and isolated from human PBMCs by magnetic activated cell sorting  using anti-FiTC microbeads  following manufacturer instructions. Then, CD4+ T cells were activated for three days with ImmunoCult Human CD3/CD28 T Cell Activator  following manufacturer instructions in the presence of 20 ng/mL IL2 . After activation, cells were expanded for 5 days in the presence of 20ng/mL IL2. Then, cells were starved of IL2 for 24 hours before stimulation with 10nM HyIL-6 in the presence or absence of different inhibitors  as indicated.Resting CD4Cells were rinsed in ice-cold PBS then lyzed in RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (ROCHE), 5 mM sodium fluoride, 2 mM sodium orthovanadate and 0.2 mM PMSF incubating on ice for 15 min. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C then protein concentrations determined using Coomassie Protein Assay Kit . For each sample, 30 μg of total protein were separated on 4%–12% Bis-Tris polyacrylamide gels  in MES SDS running buffer then blotted onto Protran 0.2 mM Nitrocellulose . Membranes were probed with 1:1000 dilution of the appropriate primary antibody anti-total-STAT3 , anti-total-RPB1 , anti-pSer2-RPB1 , anti-pSer5-RPB1  and anti-GAPDH . 1:5000 dilution of donkey anti-rabbit-HRP  or donkey anti-mouse-HRP  as the secondary antibody. Immobilon Western Chemiluminescent HRP substrate  was used for visualization.+ T cells were labeled with anti-CD4-FiTC antibody  and isolated from human PBMCs by magnetic activated cell sorting  using anti-FiTC microbeads  following manufacturer instructions. Then, CD4+ T cells were activated for three days with ImmunoCult Human CD3/CD28 T Cell Activator  following manufacturer instructions in the presence of 20 ng/mL IL2 . After activation, cells were expanded for 5 days in the presence of 20ng/mL IL2. Then, cells were starved of IL2 for 24 hours before stimulation as indicated and 105 cells were used per experiment. Cells were attached to coverslips by incubating them at 37°C for 1 hour in PBS, then PBS was replaced with RPMI supplemented with 10% FBS and cells stimulated as described. After stimulation, cells were fixed with 2% formaldehyde for 10 minutes at RT, permeabilized with ice-cold methanol for 20 minutes on ice and stained with anti-STAT3  and anti-CDK8  or anti-STAT3  and anti-CDK9  for Proximity Ligation Assays following manufacturer instructions .Resting CD4In vitro polarized human Th-1 cells generated from human PBMCs  were expanded in the presence of IL-2 for 10 days and cells were then washed with complete media and rested for 24 hr starvation in the absence of IL-2, these cells were then either not-stimulated (control) or stimulated with IL-6 or different IL-6 variants for 1 hr, cells were then immediately fixed with 1% methanol-free formaldehyde  at room temperature for 10mn with gentle rocking cells were then washed twice with cold PBS. For each STAT3 ChIP-seq library sample, approximately 10 × 106 cells were used and the fixed cell palettes were kept at −80°C prior to further processing. The ChIPseq experiments were performed as previously described  either not stimulated or stimulated with HyIL-6 in the presence or absence of 2 μM MSC2530818 variants at 37°C for 6 hr, total RNA was extracted and RNaseq libraries were prepared by Edinburg Sequencing Core facility.For RNA-seq library preparation, 5 per well in 6-well plates and transfected with siRNA SMARTPools against CDK8  and/or CDK9  24 hours later. Cells were cultured for 48 hours in the presence of the siRNA mixture before using them for FACS or western blot analysis. Knock-down efficiency was checked by western blotting using anti-CDK8 , anti-CDK9  and anti-GAPDH .CDK8 and CDK9 knock-down in HEK23T cells was done using Lipofectamine RNAiMAX following manufacturer instructions. Briefly, cell were seeded at 6x10In vitro CDKs phosphorylation reactions  were as follows: 10 μL of 2x CDK phospho assay buffer  with 100 μM ATP and 5 μL of STAT3 at 20ng/μL. Reactions were initiated by adding 5 μL of varying amounts of CDK7, CDK8 or CDK9. Reactions were incubated for 30 minutes at 30°C and stopped by adding 5 μL of 4x SDS sample buffer and heating to 95°C for 5 minutes. All kinase reactions were performed at least three times. ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo  and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).5 μL of 200 μM Alt-R CRISPR -Cas9 crRNA  were added to 5 μL of 200 μM Alt-R CRISPR-Cas9 tracrRNA , heated to 95°C for 5 min and finally the tube cooled to room temperature. Then, 1.2 μL RNA duplex was mixed with 1.7 μL Alt-R S.p. HiFi Cas9 Nuclease V3  and 2.1 μL sterile PBS and incubated at RT for 20 min. 2 × 105 Hut78 cells resuspended in 8 μL of buffer R  were added to the tubes with the RNP complexes. The electroporation parameters used were: three pulses of 1,325 V with a pulse width of 10 ms. Then, reactions were added directly into antibiotic-free media in a well of the 96-well plate and incubated at 37°C for 16 hr. HeLa cells electroporated with RNP particles were transferred into IMDM media containing 10% FCS and Pen/Strep, expanded and finally individual clones were isolated and tested for STAT3 expression levels.STAT3 KnD Hut78 cells were reconstituted with a plasmid coding for STAT3 WT or STAT3 S727A mutants by electroporation. 2 × 107 cells were resuspended in 0.25 mL of Ingenio electroporation solution , 30 μg of the appropriate construct added and the mix transferred into 4 mm gap-width cuvettes and incubated at RT for 15 minutes. Cells were electroporated using the BioRad X-Gene Pulser system , cells transferred to pre-warmed media without Pen/Strep and allow to recover for 24 hours.+ T cells isolated as described above were activated under Th-1 or Th-17 polarizing conditions. Briefly, resting human CD4+ T cells freshly isolated were activated using ImmunoCult Human CD3/CD28 T Cell Activator  following manufacturer instructions for 3 days in the presence of the cytokines required for the different CD4+ T cells populations: Th-1 (IL-2 (20 ng/ml), anti-IL-4 , IL-12 (20 ng/ml)) or Th-17 , IL-23 , anti-IL-4 , anti-IFNγ ). After three days of priming, cells were expanded in the presence of IL-2 (20 ng/ml). Th-1 and Th-17 cells were restimulated for 6 hr in the presence of PMA , Ionomycin  and Brefeldin A  before FACS analysis. In all cases cells were fixed with 2% formaldehyde and prepared to be analyzed by FACS. Cells were then permeabilised with Saponin 2% in PBS for 20 min at room temperature and then stained in Saponin 2% in PBS with the appropriate antibodies: Th-1 , anti-CD4PE , anti-CD8AF700 , anti-IFNγAF488 ) and Th-17 ) and analyzed in a CytoFLEX S (Beckman Coulter).Resting CD4MaxQuant Andromeda DAVID GO analysis tool , 2009www.bioinformatics.babraham.ac.uk)FastQC v0.11.8 Datagraph v4.5 (https://www.graphpad.com/scientific-software/prism/)PRISM v8.4.0 (GSEA v4.0.3 Metascape Bowtie v1.2.2 Samtools v1.9 bamCoverage v3.2.0 MEME Suite v5.0.2 TOMTOM BEDTools https://genome.ucsc.edu)UCSC bigWigAverageOverBed v2 (HOMER v4.10 The following software were used:Homo sapiens protein database (release date: 2018_09). Peptide and protein ratios only quantified in at least two out of the three replicates were considered, and the p values were determined by Students t test and corrected for multiple testing using the Benjamini–Hochberg procedure  using bowtie v1.2.2  with defe v5.0.2  with defy TOMTOM . STAT3 sy TOMTOM . STAT3 by TOMTOM , yieldinStatistical signinficance of differential induction of phophopeptide ratios only quantified in at least two out of the three replicates were considered, and the p values were determined by Students t test and corrected for multiple testing using the Benjamini–Hochberg procedure . For RNA


By introducing Li, the electrical and thermal transport properties were optimized collectively. The electrical transport properties were boosted remarkably via adjusting carrier concentration, and the maximum power factor (PFmax) of ~11.5 μW/cmK2 and average power factor (PFave) ~9.9 μW/cmK2 between 423 and 730 K in Pb0.99Li0.01S were achieved, which are much higher than those (~9.5 and ~7.7 μW/cmK2) of Pb0.99Na0.01S. Doping Li and Na can weaken the lattice thermal conductivity effectively. Combining the enlarged PF with suppressed total thermal conductivity, a maximum ZT ~0.5 at 730 K and a large average ZT ~0.4 at 423-730 K were obtained in p-type Pb0.99Li0.01S, which are higher than ~0.4 and ~0.3 in p-type Pb0.99Na0.01S, respectively.PbS is a latent substitute of PbTe thermoelectric materials, which is on account of its superiority in low cost and earth abundance. Here, the thermoelectric transport properties of  S2σT/κtot, where T represents absolute temperature, S expresses the Seebeck coefficient, σ denotes electrical conductivity, and κtot represents total thermal conductivity comprising electronic (κele) and lattice (κlat) contributions :(1:(10.99LnH in a reasonable range. The PFmax and PFave between 423 and 730 K of Pb0.99Li0.01S reached ~11.5 and~9.9 μW/cmK2, which are much better compared with ~9.5 and ~7.7 μW/cmK2 of Pb0.99Na0.01S. Pbx1-LixS samples possess slightly smaller κlat than that of Pbx1-NaxS because of larger mass and strain field fluctuations. At last, higher ZTmax ~0.5 at 730 K and ZTave ~0.4 at 423 K-730 K can be obtained in Pb0.99Li0.01S. The calculated thermoelectric conversion efficiency ~4.8% is achieved in Pb0.99Li0.01S with Th = 730 K and Tc = 423 K. In the future, the ZT for Li-doped PbS can also be raised through nanostructuring, manipulating band structures, and other approaches.This work indicates that Li doping is more effective than Na doping in thermoelectric performance optimization in PbS. The boosted thermoelectric performance of Li-doped PbS is completed by enhancing the PF through regulating


All of the compounds are racemic. The lactam and 3,4,5-trimethoxyphenyl rings are approximately co-planar and the orientation of the lactam and the 4-methoxyphenyl substituent is approximately orthogonal. The chiral centres, although eclipsed by geometry, have torsion angles ranging from −7.27 to 13.08° for the 3 position, and −8.69 to 13.76° for the 4 position of the β-lactam. The structures display intramolecular C—H⋯O bonding between the 3,4,5-trimethoxyphenyl ring and the lactam ketone. Further C—H⋯O interactions are observed and form either an opposing methoxy ‘buckle’ to join two molecules together or a cyclic dimer.A series of related substituted 1-azetidin-2-ones have been characterized: 3-(4-fluorophenyl)-4-(4-methoxyphenyl)-1-azetidin-2-one, C The five structurally characterized azetidin-2-ones reported herein have all been included in studies as tubulin-targeting agents with mitotic catastrophe. They have all displayed good antiproliferative effects in MCF-7 human breast-cancer cells, but tuning the substitution pattern in the aromatic ring C atoms has produced more efficacious azetidinones for further testing. The structural study of these compounds has been challenging, as the yields from synthesis were low, hence obtaining suitable crystalline samples was difficult. These structures will enable further modelling to improve the design of more effective β-lactam antibiotics.To further increase our library of β-lactam antimitotic compounds, we have investigated the systematic synthesis and activity of a range of different β-lactams based on the 1- β-lactam core –17.08 (9)° between ring plane normals  is approximately orthogonal with angles ranging from 83.59 (9) to 89.56 (8)°.It can also be seen that for both mono and di-substituted C4 lactams the angle between the lactam 1–5 are approximately eclipsed by geometry of the sp3 carbons in the lactam ring with H3—C3—C4—C18 angles ranging from 0.98° in the R isomer of 5 (di-substituted in the 4 position – more steric requirements), to a wider 13.08° in 4. The conformation at C4 is also partially eclipsed with H4—C4—C3—C26 angles of 4.96° in 3 and the largest angle of 13.76° in 4 ° plane normals for lactam N1 in PUKNUH, with the same chirality]. It can also be seen in the C2—N1—C5—C10 torsion angle of −4.3 (3) for 1 .There is a single molecule of ° Table 1. A simil2 has two independent molecules in the asymmetric unit in the monoclinic centrosymmetric system P21/c, and only one molecule is displayed in Fig. 2trans diastereomers are seen and the relative stereochemistry is 3S, 4S for the lactam with N1 and 3R, 4R for the lactam with N1A. See Table 12 show similar differences as seen above – differences in the orientation of ring B to the lactam A ring  as well as the difference in orientation the 4-methoxy group position on the B ring . The other notable difference is the orientation of the D rings to the lactam. In the N1 molecule  the A|D plane normals angle is approximately orthogonal  this A|D angle is much more acute. The twist of the group is also reflected in the torsion angles C2—C3—C26—O27 = −43.8 (3)° and C2A—C3A—C26A—O27A = 180.0 (2)°. There are no significant interactions to the furan directing this change.Compound 3, triclinic P1, see Fig. 3D ring substituent, naphthalene, also forms a hydrogen bond with the lactam ketone  β lactam. As a result of steric requirements, the 4 and 4′ substituents in both molecules show a substantial difference in A|C plane normals. Other non-bicyclic 4,4′-disubstituted β lactams are known ° and 61.454 (1)°. RIFYIO has different steric requirements with methoxycarbonylphenylethyl and acetyl groups on N1 and C3 respectively. C4 is diphenyl substituted with A|C angles of 76.79 (5)° and 66.21 (5)°.Compound s Table 1. While s1–5. The most significant are shown in Table 21 is an association with two opposing methoxy groups, see Fig. 7As well as the intramolecular hydrogen-bonding pattern described above, with the number of methoxy groups present, there are many weak C—H⋯O intermolecular interactions in 2 a ‘double buckle’ is present due to a bifurcated hydrogen bond between C12 and O11/O13 of an adjacent trimethoxyphenyl ring. These are then linked into a network via C22⋯O24A and C22A⋯O24  showing wide differences: −26.2501 (13)° in PUKNUH, −0.81417 (3)° in PUKPOD and 26.5142 (12)° in PUKPIT. From Table 11–5 is most pronounced in 5, possibly to accommodate the steric requirements of the 4,4′ disubstitution.A search of the CSD, ethyl, BAGREI; R = (methoxyphenyl)methyl, DAXKIZ; R = acetyl, GADHUO; R = phenyl, PADYAU; p = phenyl, PIHVEK, PIVHEK01; Ru = phenyl, YUDKEP. Mixed aliphatic and aromatic C4 substitution are also seen (GADJAW). There are also carboxy or cyano C4-substituted species . In all of these compounds, the group bonded to the nitrogen N1 of the lactam varies, for example from phenyl in BAGREI, 4-methyoxybenzyl in DAXKIZ and FEKRUK, 4-chlorophenyl in GADHUO, 4-nitrophenyl in IFOSIL, ethyl in POWMOD, and bis(trimethylsilyl)methyl in REBKIS.A wider search of the database for similar structural motifs to iPr substituent on the lactam N1  or ethyl (POWMOD) or incorporate a spacer such as phenylethyl, QULNUH, or (methoxycarbonyl)-2-phenylethyl) in RIFYIO, reducing the steric requirement on the lactam.However there are eight other structures with identical C4 substituents based on the core 1,3,4,4-tetramethylazetidin-2-one core. The common C4 substituent is a methyl group . However, two feature phenyl C4 substituents AHERUA and RIFYIO, and one features a dicarboxylate, QULNUH. Most feature the less bulky 1–3 uses acid activation with triphosgene with an imine , and 5 acetic acid and N-(4-methoxybenzylidene)-3,4,5-trimethoxybenzenamine. White crystalline solid. Yield 7.5%,2: 2-(Furan-2-yl)acetic acid and N-(4-methoxybenzylidene)-3,4,5-trimethoxybenzenamine. Brown crystals. Yield 4.9%.3: 2-acetic acid and N-(4-methoxybenzylidene)-3,4,5-trimethoxybenzenamine. Pale-yellow crystalline powder. Yield 6.9%.4: 2-acetyl chloride and N-(4-methoxybenzylidene)-3,4,5-trimethoxybenzenamine. White powder. Yield 1.1%.5: Phenyl acetyl chloride and bis-(4-methoxyphenyl)methanone. White powder. Yield 17%.The crude product was purified by flash column chromatography over silica gel (eluent: hexane/ethyl acetate gradient). The eluent was evaporated and compounds were recrystallized from ethanol.Uiso(H) = 1.2Ueq(C)  or 1.5Ueq(C) (methyl). Compounds 2 and 5 were refined with extinction, 0.0011 (2) and 0.00057 (14) respectively.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020008555/zl2789sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, 4, 5, global. DOI: 10.1107/S2056989020008555/zl27891sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989020008555/zl27892sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989020008555/zl27893sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989020008555/zl27894sup5.hklStructure factors: contains datablock(s) 4. DOI: 10.1107/S2056989020008555/zl27895sup6.hklStructure factors: contains datablock(s) 5. DOI: Click here for additional data file.10.1107/S2056989020008555/zl27891sup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008555/zl27892sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008555/zl27893sup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008555/zl27894sup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008555/zl27895sup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008555/zl2789sup12.tifOverlay of 1 and PUKNUH with overlay centres N1 C3 and O17/O3 and all oxygen atoms labelled, showing the difference in the B ring orientation. The para-methoxy substituent is also flipped. The C and D ring orientations are similar. DOI: Click here for additional data file.10.1107/S2056989020008555/zl2789sup13.tifOverlay with inversion of each unique molecule in the asymmetric unit of 2, showing the difference in the D ring orientations and para-methoxy groups as well as the rotation of the D ring (furan). DOI: Click here for additional data file.10.1107/S2056989020008555/zl2789sup14.tifOverlay with inversion of each unique molecule in the asymmetric unit of 5, showing the difference in the B ring orientations, and C' ring para-methoxy group differences. DOI: 2012288, 2012287, 2012286, 2012285, 2012284CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The phenyl and triazole rings are almost perpendicular to each other, forming a dihedral angle of 89.5 (2)°. In the crystal, the molecules are linked by weak C—H⋯π(phenyl) interactions, forming supramolecular chains extending along the c-axis direction. The crystal packing is further consolidated by intermolecular N—H⋯S hydrogen bonds and by weak C—H⋯S interactions, yielding double chains propagating along the a-axis direction. The crystal studied was refined as a racemic twin.In the title compound, C Two red spots are present in close proximity to the S and N—H atoms involved in hydrogen bonding. As expected, the absence of red and blue triangles on the shape-index surface and the small, flat segments delineated by the blue line in the surface mapped over curvedness indicate the absence of π–π stacking interactions in the crystal structure, while the red regions over the shape-index surface are due to the presence of C—H⋯π interactions.In order to investigate the intermolecular interactions in the structure of ss Fig. 3. In the I provide some quantitative information about the individual contributions of the intermolecular interactions in the asymmetric unit  and S⋯H/H⋯S (14.6%) intermolecular contacts. This indicates that van der Waals forces have an important influence on the consolidation of the crystal structure. The other contacts contribute less to the Hirshfeld surfaces: F⋯H/H⋯F (11%), Br⋯H/H⋯Br (9.8%), H⋯C/C⋯H (8.4%), N⋯H/H⋯N (7.5%), Br⋯C/C⋯Br (3.5%), S⋯N/N⋯S (1%), Br⋯Br (0.5%) and C⋯S/S⋯C (0.5%).The two-dimensional fingerprint maps for t Figs. 4 and 5 ▸;et al., 2016H)-thione derivatives gave six hits containing a substituted triazole ring, namely: 3-(adamantan-1-yl)-4-benzyl-1H-1,2,4-triazole-5(4H)-thione -thione -4-(prop-2-en-1-yl)-1H-1,2,4-triazole-5(4H)-thione -thione -thione -4-phenyl-2,4-dihydro-1,2,4-triazole-3-thione -1-(2-bromo-4-fluorophenyl)-2-thiosemicarbazide C, which was then cyclized to the title compound I by heating in aqueous sodium hydroxide as outlined in Fig. 6All chemicals and solvents were used as purchased without further purification. The melting point was determined using an electrothermal digital melting-point apparatus and uncorrected. The NMR spectra were recorded at room temperature in DMSO-in vacuo and an aqueous sodium hydroxide solution  was added to the residue and the mixture was heated under reflux for 4 h, then filtered hot. On cooling, the mixture was acidified with hydrochloric acid (pH 1–2) and the precipitated crude product was filtered, washed with water, dried and crystallized from an aqueous medium to yield 3.06 g (75%) of the title compound (C18H19BrFN3S) as fine colourless crystals (m.p. 577–579 K). Single crystals suitable for X-ray diffraction were obtained by slow evaporation of a solution of the title compound in EtOH/CHCl3  at room temperature. 1H NMR : δ 1.47–1.71 , 1.87–1.89 , 7.47–7.52 , 7.69–7.71 , 7.87–7.90 , 13.86 . 13C NMR : δ 27.92, 36.08, 36.57, 38.49 (adamantane-C), 115.0, 121.15, 131.65, 134.16, 135.21, 161.86 (Ar-C), 159.46 (triazole C=N), 169.41 (triazole C=S).2-Bromo-4-fluorophenyl isothiocyanate  was added to a solution of adamantane-1-carbohydrazide , in ethanol (10 mL), and the mixture was heated under reflux with stirring for one h. Ethanol was then distilled off Uiso(H) set to 1.2 to 1.5Ueq. The structure was refined as a racemic twin [BASF: 0.50 (2)]. Four reflections  global, I. DOI: 10.1107/S2056989020000092/xi2020Isup2.hklStructure factors: contains datablock(s) I. DOI: 1975689CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A new nickel(II) complex of a hexaazamacrocycle containing 4-flurophenethyl pendant arms was synthesized by metal template condensation in a one-pot reaction of formaldehyde and amines in the presence of nickel(II) ions and its X-ray crystal structure was determined. 24H36F2N6)](ClO4)2 or [NiL](ClO4)2 -1,3,5,8,10,12-hexaazacyclotetradecane) was synthesized by a one-pot reaction of template condensation and its X-ray crystal structure was determined. The nickel(II) ion lies close by a twofold axis and the complex displays whole-molecule disorder. Ligand L, a hexaazacyclotetradecane ring having 4-fluorophenethyl side chains attached to uncoordinated nitrogen atoms, adopts a trans III  configuration. The average Ni—N bond distance is 1.934 (9) Å, which is quite similar to those of other nickel(II) complexes with similar ligands. The nickel(II) ion is located 0.051 (7) Å above the least-squares plane through the four coordinated N atoms. The average C—N bond distance and C—N—C angle involving uncoordinated nitrogen atoms are 1.425 (12) Å and 118.0 (9)°, respectively, indicating a significant contribution of sp2 hybridization for these N atoms. The intermolecular N—H⋯O, C—H⋯O/F hydrogen bonds of the complex form a network structure, which looks like a seamless floral lace pattern.The square-planar nickel(II) title complex, [Ni(C It often produces new macrocyclic complexes in one-pot reactions with high yield II ion lies close by a special position (twofold axis) and the [NiL]2+ complex occurs in two orientations with fixed occupancies of 0.50. The refinement of this whole-molecule disorder needed additional restraints (see Refinement section). The occupancies of the disordered perchlorate ion are 0.795 (7) and 0.205 (7). The nickel(II) ion is coordinated to the four nitrogens N2, N3, N2′ and N3′, and the complex has a square-planar coordination geometry. The 14-membered ring skeleton adopts the thermodynamically most stable trans-III configuration with R,R,S,S chirality of the four coordinated nitrogen atoms  of the 14-membered 1,3,5,8,10,12-hexaazacyclotetradecane ring skeleton. The 4-fluorophenethyl pendants are positioned above and below the square coordination plane. The six-membered chelate rings adopt a chair conformation and the five-membered chelate rings assume a gauche conformation.The molecular structure of the title compound is shown in Fig. 1et al., 1999et al., 1998et al., 1996sp2 hybridization of the bridgehead nitrogen atoms (N1 and N1′)  as well as C—H⋯A (A = O or F) hydrogen bonds in the crystal packing of [NiL](ClO4)2. Hydrogen-bonding interactions between N—H or C—H groups of the ligand L and perchlorate oxygen atoms are summarized in Table 2A of a neighboring molecule  Å and C4′—H4′A⋯F1′ = 128.4 (8)°]. These interactions form a chain structure extending in the .Crystal data, data collection and structure refinement details are summarized in Table 3SHELXL: (1) occupancy factors were set at 0.50, (2) the two chemically equivalent halves of the complex were restrained to be similar using the ‘SAME’ command, (3) the fluorinated benzene rings were given a weak ‘FLAT’ restraint, (4) Ni1 required a strong ‘ISOR’ restraint and (5) displacement factors for atom pairs related about the special position were constrained to be equal (EADP).The refinement of the whole-molecule disorder employed the following constraints and restraints in The perchlorate anion is disordered over two sets of atomic sites with occupancy ratios of 0.795 (7):0.205 (7).10.1107/S2056989020016795/vm2242sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020016795/vm2242Isup2.hklStructure factors: contains datablock(s) I. DOI: 2053166CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PIbisphosphate P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid–containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PIP2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PIP2 cycle.Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid–containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor–dependent cell proliferation, type 2 diabetes, and obsessive–compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity  In a C (PLC) , 12, 13. mammals , 14, 15, mammals .et al. (via a homology search for the sequences encoding the membrane proteins containing active site motifs common to PAP2/LPP. The SMS1 and SMS2 proteins generate DG and SM via the transfer of phosphocholine from PC to ceramide (via the transfer of phosphoethanolamine from PE to ceramide (Sphingomyelin synthase (SMS) is a DG-generating enzyme , 18. Theet al.  identificeramide . SMSr isceramide    , 23, 24, (SAMDs) . Moreove (SAMDs) . In part (SAMDs) , were sivia hydrolysis of PA, PI, PE, and PC in the absence of ceramide. The PAP, PI-phospholipase C (PLC), PE-PLC, and PC-PLC activities were much stronger than the CPES activity . Intriguingly, SMSr exhibited a substrate specificity for SFA- and/or MUFA-containing PA and PC molecular species, but not polyunsaturated fatty acids (PUFA)-containing PA and PC. Taken together, we revealed the previously unrecognized enzyme activities of mammalian SMSr, novel PAP, PI-PLC, PE-PLC, and PC-PLC activities, to produce SFA and/or MUFA-containing DG molecular species independent of ceramide in the ER membrane.Taken together, SMSr can supply DG to DGKδ , whereasA). SMSr-ΔSAMD showed no significant difference in DG production levels compared with FL-SMSr . However, the SMSr-ΔSAMD yield was approximately six-fold higher than that of FL-SMSr (data not shown). Therefore, we employed SMSr-ΔSAMD for further investigation.To determine whether SMSr possesses other DG-generating activity in addition to CPES activity, we first expressed N-terminal hexahistidine (6×His)-tagged human full-length (FL) SMSr and SMSr-ΔSAMD, which lacks an oligomer formation domain, SAMD , 33, in Fig. 1A). Thus, the purified 6× His-SMSr-ΔSAMD is likely to be a monomer because its calculated molecular mass is 40.8 kDa (SMSr-ΔSAMD: 39.2 kDa +6×His: 1.6 kDa). Immunoblotting and silver staining showed that a single protein band at approximately 40 kDa was detected (B), indicating that 6× His-SMSr-ΔSAMD was obtained with a high purity. The yield was ∼6 μg per 1 L of an Sf9 cell culture. We confirmed that the purified SMSr-ΔSAMD slightly produced DG in the presence of PE and ceramide micelles (C). However, the DG-generating activity was exceedingly low (∼3 pmol/mg/min). Moreover, only trace amounts of CPE were generated by the purified SMSr-ΔSAMD (D).Next, to further purify 6× His-SMSr-ΔSAMD, size-exclusion chromatography was conducted after Ni-affinity chromatography. SMSr-ΔSAMD was eluted as a single peak in a volume of 13.9 ml (∼56.1 kDa) A. Thus, detected B, indicamicelles C. HoweveSr-ΔSAMD D.Figure in vitro by incubating the substrate micelle mixture followed by quantitation of the produced DG levels using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (A). Compared with the DG-generating activity in the presence of ceramide and 16:0/18:1-PE (substrates for CPES), SMSr showed a much more intensive PAP activity (∼340-fold), even in the absence of ceramide. The enzyme also has stronger PI-PLC (∼12-fold), PE-PLC (∼4-fold), PC-PLC (∼3-fold), and PG-PLC (∼1.5-fold) activities. However, the PS hydrolysis activity of SMSr was not detectable. To further investigate the selectivity of SMSr for phosphoinositides, we compared the hydrolysis activities of purified SMSr-ΔSAMD against 18:0/20:4-PI and 18:0/20:4-PIP2 (B). The enzyme showed a ten-fold stronger PI-PLC activity than the PIP2-PLC activity. This result indicates that the catalytic selectivity for the phosphoinositides of SMSr is opposite to that of mammalian PI-PLC P2-PLC), which hydrolyzes PIP2 much more strongly than PI P2:PI = 50:1)  of purified SMSr-ΔSAMD against various phospholipids -PA, 16:0/18:1-PC, 16:0/18:1-PE, 16:0/18:1-PG, 16:0/18:1-PI and 16:0/18:1-PS) C-MS/MS) , 34  A. ComparIP2 B. The en = 50:1)  35).Fig.Figin vi2-PLC, and PG-PLC activities. Next, a kinetic analysis of the enzyme was performed using n-dodecyl-β-D-maltoside (DDM)/Cholesteryl hemisuccinate (CHS)/phospholipid-mixed micelles. The Km value for 16:0/18:1-PA was 3.92 ± 0.536 mol% (Vmax: 704.6 ± 46 pmol/mg/min) (C), and the value for 16:0/18:1-PI was 6.83 ± 2.076 (mol%) (Vmax: 32.4 ± 5.6 pmol/mg/min) (D). The Km value and Vmax for 16:0/18:1-PA are comparable with those of LPPs  C, and th/mg/min) D. The Km of LPPs  (Table 2A), we assumed that ceramide affects DG-generating activity of SMSr-ΔSAMD. Thus, the effects of d18:1/18:0-ceramide on hydrolysis activities of SMSr-ΔSAMD for other glycerophospholipids  were determined (A). However, ceramide had no detectable effects on hydrolysis of PA, PI, and PC (B–D), suggesting that the inhibitory effect of ceramide is PE selective.Because DG production activity by SMSr-ΔSAMD in the presence of d18:1/18:0-ceramide and PE was lower than that in presence of PE alone A, we asstermined . We conftermined A. Howeve, and PC , B–D, suA and B). SMSr was intensively active against SFA and/or MUFA-containing PA species, such as 16:0/18:1-, 16:0/16:0-, and 18:1/18:1-PA. However, SMSr exhibited relatively low activities against PUFA-containing PA, such as 18:0/20:4-PA, which is exclusively derived from the PIP2 cycle . The PAP and PC-PLC activities were significantly inhibited by 1 mM propranolol  and by 500 μM D609  (C and E). However, the inhibitors had no significant effects on the PI-PLC activity (D).We examined the effects of propranolol, which is a well-known PAP inhibitor , 39, andctively) , C and Eactivity D.A), and PAP, PI-PLC, and CPES activities in the immunoprecipitates were measured (B–D). The SMSr expressed in the COS-7 cells exhibited PAP and PI-PLC activities (B and C). However, a CPE synthase-inactive mutant of SMSr (D348E) (D), suggesting that human SMSr lacks the ability to synthesize considerable amounts of CPE, as Vacaru et al. reported   failed t (D348E) D, suggesreported .Figure 6A). The total PA levels in COS-7 cells overexpressing SMSr-V5 were significantly decreased . This result suggests that SMSr produced DG, at least in part, by hydrolyzing PA in mammalian cells.We previously reported that SMSr expressed in COS-7 cells generated DG . To deteFig. 72-PLC . The purified enzyme gave almost a single band with a molecular mass of 43,000 via SDS gel electrophoresis. It is likely that size-exclusion chromatography with a buffer containing a nonionic, nondenaturing detergent, Nonidet P-40 (NP-40), at critical micelle concentration (0.018%) was critical for the successful purification.The purification of six-transmembrane proteins, SMS and SMSr, is generally difficult . Howevertography , A and Bin vitro (A and B). Interestingly, compared with the DG-generating activity in the presence of PE and ceramide (substrates of CPE synthase), purified human SMSr showed a ∼340-fold stronger PAP activity, even in the absence of ceramide. Considering the report that SMSr metabolized approximately 300-fold less ceramide than SMS1  (C and D), which are almost the same as those of other PAPs, LPP1 (PAP2a) (B and C). Therefore, SMSr probably shows CPES, PAP, PE-PLC, PI-PLC, PC-PLC, and PG-PLC activities through the same catalytic site.Using the highly purified enzyme, we demonstrated for the first time that SMSr displayed not only CPES activity but also PAP, PE-PLC, PI-PLC, PC-PLC, and PG-PLC activities in vitro , A and Bhan SMS1  (Table 183 mol%) , C and D (PAP2a) , and lip (PAP2a)  (Table 2 (PAP2a) , 20, 47, (PAP2a) , B and CA), but not its PAP, PI-PLC, or PC-PLC activity (B–D). It is possible that PE-PLC activity of SMSr is competitive with CPE synthase activity of SMSr. SMSr has CPE synthase activity and, consequently, ceramide can occupy the substrate (ceramide/PE) binding site of SMSr  of SMSr at least in part.In this study, we found that ceramide selectively affected PE-PLC activity of SMSr-ΔSAMD A, but noactivity , B–D. It of SMSr . Therefo of SMSr , 48. TakA). However, two lines of evidence support that the PI-PLC activity of SMSr is comparable with its PAP activity in cells. First, the amount of PI, PC, and PE in the ER membrane was more than 20-fold, 100-fold, and 40-fold higher than that of PA, respectively   the PAP activity (B and C). These results suggest that the PI-PLC activity of SMSr expressed in mammalian cells is comparable with its PAP activity. Therefore, PAP and PI-PLC (and PE-PLC and PC-PLC) are likely to represent the substantial activity of SMSr.The PI-PLC, PE-PLC, and PC-PLC activities of purified SMSr were 28∼100-fold weaker than the PAP activity A. Howevectively)  49, 50), 50A). Hactivity , B and C2) . LPPs contain six transmembrane domains and are primarily localized to the plasma membrane  see ? LPPs ha2) , 14, 51., and PC , A and Bmembrane , 51. It membrane , 53. SMSmembrane , 33. Aspmembrane . It is pA and B). This unique selectivity reinforces the hypothesis that SMSr and its downstream enzyme, DGKδ, are components of the alternative DG/PA-signaling pathway, which is independent of the PIP2 cycle  . On the P2 cycle .2-PLC? We found that SMSr also hydrolyzed 18:0/20:4-PIP2in vitro, and this activity was approximately tenfold weaker than the 18:0/20:4-PI-PLC activity (B). Notably, although mammalian PIP2-PLC  also hydrolyzes both PIP2 and PI, its PIP2-PLC activity is 30∼50-fold stronger than the PI-PLC activity P2 levels in the mammalian cells are ∼100-fold lower than the PI levels P2 because little or no 18:0/20:4-PIP2 exists in the ER membrane.What are the differences between SMSr as PI-PLC and PIPactivity B. Notablactivity , 54. TheI levels . MoreoveI levels , whereasmembrane , 58. TakE). Therefore, it is likely that SMSr is PC-PLC, which has been awaited for molecular entity identification for quite some time (∼40 years).It has been suggested that PC-specific phospholipase C (PC-PLC) plays pivotal roles in signal transduction pathways that are involved in atherogenesis, inflammation, and carcinogenesis , 40, 59.Fig. 5PE-PLC activity in mammalian cells was discovered approximately 30 years ago , 46, 60 2 cycle under high-glucose stimulation P2 cycle becomes clearer.We previously reported that in C2C12 myoblasts, DGKδ apparently prefers to phosphorylate palmitic acid (16:0) and/or palmitoleic acid (16:1)-containing DG molecular species, but not 20:4-containing DG delivered from the PIPmulation . Howevere enzyme . Howeverin vitro, in addition to CPES activity. In particular, PAP (∼300-fold), PI-PLC (∼10-fold), PC-PLC (∼3-fold), and PE-PLC activity (∼4-fold) were stronger than the CPES activity. Thus, beyond the enzyme’s previously established CPES activity, SMSr can be categorized as a new type of PLC with a broad head group specificity, i.e., a multi-glycerophospholipid PLC hydrolase (MG-PLC). The current results allow us to propose the new DG/PA metabolic pathway “Luminal PA, PI, PC and PE → SMSr (MG-PLC) → DG → flip-flop → DGKδ → cytoplasmic PA” in the ER membrane, which is independent of the PIP2 cycle , 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0/18:1-PS), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1/18:1-PA), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol (18:0/20:4-PI), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- P2), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate (18:0/20:4-PA), and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (18:0/22:6-PA) were obtained from Avanti Polar Lipids. 1,2-dimyristoyl-sn-glycerol (14:0/14:0-DG), 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (16:0/18:1-PG), and N-stearoyl-D-erythro-sphingosine (d18:1/18:0-Cer) were obtained from Cayman Chemical Company. 16:0/18:1-sn-glycero-3-phosphocholine (16:0/18:1-PC) was purchased from Sigma-Aldrich.Lipids: 1,2-dipalmitoyl-sn-glycero-3-phosphate (16:0/16:0-PA), 1-palmitoyl-2-oleoyl-Detergents: n-dodecyl-β-D-maltoside (DDM) was obtained from Cayman Chemical Company. Cholesteryl hemisuccinate (CHS) was purchased from Sigma-Aldrich.Human SMSr and SMSr-ΔSAMD (amino acid residues 79–413) tagged with N-terminal 6×His or C-terminal V5 were previously made . 6× His-Rabbit polyclonal anti-His-tag antibody (PM032) was obtained from Medical and Biological Laboratories. Mouse monoclonal anti-V5 antibody  was obtained from Thermo Fisher Scientific. A peroxidase-conjugated goat anti-mouse IgG antibody was purchased from Bethyl Laboratories. A peroxidase-conjugated goat anti-rabbit IgG antibody (111-036-045) was obtained from Jackson Immuno Research.2 as described  according to the manufacturer’s instruction manual. After transfection, the cells were cultured for an additional 24 h and were used for the experiments.COS-7 cells were maintained on 150-mm dishes (Thermo Fisher Scientific) in Dulbecco’s Modified Eagle’s Medium (DMEM)  containing 10% fetal bovine serum (Thermo Fisher Scientific), 100 units/ml penicillin G , and 100 μg/ml streptomycin  at 37 °C in an atmosphere containing 5% COescribed . For qua2 in the dark. Volume of the medium was kept at 20 to 30% of flask volume.Sf9 cells were maintained in Sf-900 II serum-free medium (Invitrogen) in sterile Erlenmeyer flask at 120 rpm and 28 °C without COhttps://www.mirusbio.com/assets/protocols/flashbac-baculovirus-expression-systems-protocol.pdf) (0), 1.0 × 106 cells were plated on 35-mm dishes. The DNAs (flashBAC and pOET3 vector) were transfected using TransIT-Insect Reagent (Mirus Bio) according to the manufacturer’s instruction manual. The baculoviral stocks  were harvested at 5 days posttransfection supernatants and passed through a 0.22 μm filter.To generate recombinant baculovirus, the flashBAC system (Oxford Expression Technologies) and the pOET3 vector were used as described in flashBAC System Transfection Protocol (col.pdf) , 62. For6 cells/ml, multiplicity of infection (MOI) of 0.1. Viral titer (plaque forming unit (pfu)/ml) was determined via plaque assay as described in flashBAC System Transfection Protocol. Viral stocks were stored at −80 °C until use.Baculoviral stocks were amplified for seven rounds of amplification at 2 × 106 cells/ml and infected cells with recombinant baculovirus at MOI of 2. Infected cells were harvested after 24 h and the pellets were resuspended in 40% (v/v) glycerol diluted in phosphate-buffered saline. The cell samples were flash-frozen in liquid nitrogen and stored at −80 °C until use.To express recombinant proteins, Sf9 cells were seeded at 3 × 10via the protocol described by JCIMPT, an NIH Structural Biology RoadMap Initiative Project (https://commonfund.nih.gov/sites/default/files/JCIMPT_MembranePrep.pdf) (The membranes of Sf9 cells containing overexpressed proteins were prepared rep.pdf) , 64, 65.g for 30 min at 4 °C, cell pellets were resuspended in ice-cold hypotonic buffer (10 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 20 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin, 20 μg/ml leupeptin, 20 μg/ml pepstatin and 20 μg/ml soybean trypsin inhibitor), and cells were homogenized on ice via homogenizer. After ultracentrifugation at 100,000g for 30 min at 4 °C, pellets were resuspended in ice-cold high-osmolarity buffer (10 mM HEPES-NaOH (pH 7.5), 1 M NaCl, 10 mM MgCl2, 20 mM KCl, 1 mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 20 μg/ml pepstatin, and 20 μg/ml soybean trypsin inhibitor) and homogenized on ice via homogenizer. This procedure using high-osmolarity buffer was repeated twice. After ultracentrifugation at 100,000g for 30 min at 4 °C, Sf9 membranes were resuspended in 40% (v/v) glycerol diluted in high-osmolarity buffer and homogenized. The Sf9 membranes were flash-frozen in liquid nitrogen and stored at −80 °C until use.Frozen Sf9 cells expressing recombinant proteins were thawed at 4 °C. After centrifugation at 10,000via affinity chromatography. Frozen Sf9 membranes containing recombinant proteins were thawed at 4 °C. After ultracentrifugation at 100,000g for 30 min at 4 °C, the purified membranes were lysed via homogenization on ice with ice-cold lysis buffer  glycerol, 1% (v/v) NP-40, 20 mM imidazole, 1 mM PMSF, 0.1 mM DTT, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 20 μg/ml pepstatin, and 20 μg/ml soybean trypsin inhibitor) and incubated at 4 °C for 1 h with gently stirring via magnetic stirrer. The supernatant (1% NP-40 soluble fraction) was isolated by ultracentrifugation at 200,000g for 30 min at 4 °C, and then the supernatant was incubated with nickel–nitrilotriacetic acid (Ni-NTA) agarose  at 4 °C for 2 h with gently rotation. After centrifugation at 200g for 10 min at 4 °C, the Ni-NTA agarose was resuspended in lysis buffer and transferred into an empty column. The beads were washed with 30 ml of lysis buffer, followed by washing with 10 ml of wash buffer  glycerol, 0.5% (v/v) NP-40, 50 mM imidazole, 0.1 mM DTT). Subsequently, the bound proteins were eluted with an elution buffer  glycerol, 0.1% (v/v) NP-40, 500 mM imidazole, 0.1 mM DTT). The partially purified proteins were further purified via size-exclusion chromatography. After concentration using Amicon Ultra-15 (Merk), the partially purified samples were subjected to size-exclusion chromatography on an ENrich SEC 650 column (Bio-Rad equipped with a Bio-Rad NGC chromatography system). The column was equilibrated with 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 5% glycerol, 0.5 mM dithiothreitol, and 0.018% NP-40.The 6× His-tagged proteins were partially purified The purified proteins were mixed with a 5×SDS sample buffer and incubated at 37 °C for 2 h instead of boiling. The samples were analyzed by SDS-PAGE and an immunoblot analysis, as described .To prepare detergents stock solution (10% (w/v) n-dodecyl-β-D-maltoside (DDM) and 2% (w/v) cholesteryl hemisuccinate (CHS)), 5 g of DDM was added into 40 ml of 200 mM Tris-HCl (pH 8.0), followed by gently rotation until DDM goes into solution. The detergent (1 g of CHS) was added to DDM solution and sonicated until solution becomes translucent. After sonication, 10 ml of 200 mM Tris-HCl (pH 8.0) was added and incubated at 25 °C with gently rotation until the solution becomes transparent. The detergents solution was stored at −25 °C until use.2 gas to yield a lipid film on the wall of the vial. The lipid film was resuspended in the 2×reaction buffer (40 mM Tris-HCl (pH 7.4), 30 mM KCl, 200 mM NaCl, 600 mM sucrose, 0.2% (w/v) DDM, 0.04% (w/v) CHS) to a final total lipid concentration of 0.1 mM each. The 2×reaction solutions containing lipids were vortexed for 3 min and sonicated in a bath sonicator (Sonifilter model 450) four times for 2 min at 65 °C.The reaction solution for PLC and CPES activity assay was prepared as we reported . Phosphovia a DDM mixed micellar phospholipase C assay previously published  of purified proteins were evaluated ublished , 67 withSMSr-containing samples (10 μl) were diluted in 10 μl ice-cold 2×reaction buffer on ice , 15 mM KCl, 200 mM NaCl, 300 mM sucrose, 0.1% (w/v) DDM, 0.02% CHS, and 50 μM phospholipids ). For a CPE synthase activity assay, d18:1/18:0-ceramide was added into the reaction buffer . Because SMSr is membrane-bound and lipid-dependent enzyme, the surface dilution kinetic scheme was used , 69. TheAfter incubation (the enzyme reaction) for 2 h at 37 °C, lipids were extracted by Bligh & Dyer method , followet-test or one-way ANOVA using GraphPad Prism 8 (GraphPad).The data are represented as the means ± SD and were analyzed using Student’s All data are contained within the article.The authors declare no conflicts of interest associated with the contents of this article.


Scientific Reports 10.1038/s41598-020-71814-0, published online 10 September 2020Correction to: This Article contains an error in the y-axis label of Figure 5, where the unit of measurement is incorrect.2 (mmol m-1 day-1)” should read: “FCO2 (mmol m-2 day-1)”“FCOThe correct Figure 5 appears below as Figure