Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in  •Xenopus embryos on up to 13 conserved SP/TP sites.E47 is phosphorylated in •In vitro, E47 protein can be phosphorylated by Cyclin-dependent-kinases.•Under-phosphorylated E47 enhances activity of 3 bHLH reprogramming factors.•Under-phosphorylated E47 enhances neuronal differentiation by hetero-dimerisation.•E47 phospho-status alters chromatin binding but not overall protein stability. Tissue  in vivo . Recentl in vivo .Drosophila daughterless and mammalian HEB, E2-2, E12 and E47 [Class II bHLH proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins such as  and E47 ,11. ThusXenopus frog embryos as a model system.Analogous to potential phosphorylation sites described in class II bHLH factors, E proteins also have multiple conserved Serine/Threonine Proline (SP/TP) sites, and phospho-regulation of these sites on a tissue-specific or global level has the potential to regulate differentiation across multiple lineages. To our knowledge, only one (S140) out of thirteen potential SP/TP sites in E47 has previously been ascribed a specific regulatory role in muscle differentiation . We ther22.1NM_011548.4) was cloned into pCS2 by In-fusion HD (Clonetec/Takara). 13T/S-A and 12T/S-A E47 were generated by QuikChange Site Directed Mutagenesis (Agilent). A single C terminal HA tag was added. WT mouse Ascl1, Ngn2 and MyoD, and 6S-A mouse Ascl1 have been described previously [Wild-type (WT) mouse E47  Act, 1986 and associated guidelines. A description of experiments using ARRIVE guidelines is provided in Ref. . Acquisi2.3Whole embryo RNA was extracted, cDNAs prepared and qPCR conducted as described in Refs. ,7.2.4In vitro kinase assay was conducted as described [escribed . Proteinescribed , and allescribed .2.5EF1α, and mRNA levels in injected embryos were calculated relative to stage-matched uninjected controls. Mean values and the standard error of the mean (s.e.m.) were calculated from N independent experiments. Statistical significance was determined using a paired two-tailed Student's t-test with not significant = NS; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For ISH data, experiments were conducted in independent duplicate or triplicate and the N numbers refer to the range of total numbers of embryos in each injection category.For western blotting, experiments were performed in independent triplicate with representative results shown. For qPCR data, mRNA expression was normalised to housekeeping gene 33.1Xenopus as a flexible in vivo system to test whether the ability of E proteins to drive cellular differentiation is regulated by multi-site phosphorylation on Serine/Threonine-Proline sites during early embryogenesis.Multi-site phosphorylation of class II bHLH transcription factors by Cyclin-dependent kinases (Cdks) has been demonstrated to limit their ability to drive differentiation and reprogramming of cells in all three embryonic germ layers . While lXenopus embryos and protein extracts were prepared at stage 11. Western blotting (in vivo in developing Xenopus embryos on multiple SP/TP sites.Firstly, we generated constructs encoding either WT E47 or 13T/S-A E47, where all of the potential SP/TP sites in E47 are mutated to Alanine-Proline A. To detblotting B revealsblotting C, WT E47in vitro recombinant kinase assays to phosphorylate E47 protein translated in reticulocyte lysate, and separating proteins on Phos-tag™ gel to more easily identify phosphorylated forms (Class II bHLH transcription factors can be phosphorylated on SP/TP sites by Cdks ,5,8. To ed forms D. Some mXenopus embryos, while forced expression of MyoD expands myogenesis in the mesoderm [E proteins are traditionally viewed as the heterodimeric partners that work with class II bHLH transcription factors to drive neurogenesis or myogenesis ,15. We nmesoderm ,5,7. Phomesoderm , so an aWT and phospho-mutant E47 mRNAs were co-injected with WT Ascl1 A, Ngn2 B, or MyoXenopus ectoderm into neurons (Preventing phosphorylation of several class II bHLH proteins by mutating SP and TP sites results in their enhanced activity , and usi neurons D. At thi3.2Xenopus, while E proteins alone fail to upregulate neural genes in either whole embryos or explant assays [in vivo through both an expansion of the progenitor domain and subsequent neuronal differentiation.Class II bHLH proneural transcription factors are thought to act as hetero-dimers with E proteins to drive ectopic neurogenesis in t assays . To detet assays . While WWhile E47 has been described to act through hetero-dimerisation in neurogenesis , E47 has3.3Finally, we explored the mechanisms by which phosphorylation might regulate the activity of E47. Preventing multi-site phosphorylation of class II bHLH proteins has been shown to increase protein stability . To deteXenopus embryos followed by western blotting to detect E47 protein in cytoplasm and bound to chromatin (We have also previously seen that preventing multi-site phosphorylation can enhance chromatin binding of class II bHLH proteins . To detehromatin C+D. Inte4Xenopus embryos on up to 13 conserved SP/TP sites and mutation of these sites enhances both endogenous neurogenesis and differentiation induced by ectopic class II bHLH reprogramming factors. E47 is phosphorylated in vitro by CyclinA/Cdk2 and CyclinB/Cdk1, suggesting potential regulation during the G2/M phase of the cell cycle, although we note that there is no measure of relative kinase activity against another independent substrate making it hard to draw firm conclusions about kinase specificity from this experiment. We would however note that a similar kinase assay using the class II bHLH proneural protein Neurogenin3 demonstrates phosphorylation by all four Cyclin/Cdk combinations [We find that E47 is phosphorylated in inations , consistXenopus embryos. We note that Drosophila homologue daughterless has a long half-life of over 4 h during neurogenesis [Xenopus embryos; an effect that requires hetero-dimerisation with endogenous factors. Thus, multi-site phosphorylation controls the activity of classes I, II and VI bHLH transcription factors [Class II bHLH transcription factors typically have short half-lives of around 20 min, while preventing their multi-site phosphorylation results in significant protein stabilisation ,21. In cogenesis , suggest factors , represeXenopus ectoderm compared to WT Ascl1 [Xenopus by supplying extra WT or phospho-mutant E47 along with 6S-A Ascl1 (In the field of mammalian cellular reprogramming, class II bHLH proteins such as Ascl1 are routinely utilised for their potent ability to direct cell-type specific differentiation, but advances in our understanding of the control of the reprogramming process is needed to improve both the conversion efficiency and maturity of induced cells. Phospho-mutant 6S-A Ascl1 shows an enhanced ability to convert mammalian fibroblasts into mature neurons compared to the wild-type protein, and generates more ectopic neurons in WT Ascl1 ,23. Howe-A Ascl1 D. 6S-A Ain vitro cell reprogramming.Taken together, our data suggest that cycle lengthening and exit during development may result in de-phosphorylation of both class I and class II components of the hetero-dimeric bHLH complex, and both components may contribute to enhancement of differentiation. Furthermore, our data suggest that using forms of class II or class I bHLH proteins that cannot be phosphorylated by Cdks may enhance


Saponification is the process in which triglycerides are combined with a strong base to form fatty acid metal salts during the soap-making process. The distribution of unsaturated and saturated fatty acid determines the hardness, aroma, cleansing, lather, and moisturizing abilities of soaps. Plant extracts, such as rosemary, vegetable, and essential oils are frequently added to soaps to enhance quality and sensory appeal. Three natural soaps were formulated using cold saponification to produce a base or control bar (BB), hibiscus rosehip bar (H), and a forest grove bar (FG). Rosemary extract (R) or essential oil (A) blends were added as additives to each formulation prior to curing to evaluate the effects of natural plant additives on the lipid composition and sensory characteristics of these natural herbal soaps. A total of seven natural soaps, three without additives  and four with additives , were manufactured and studied. The majority (86–99%) of the polyunsaturated fatty acids (5.0–7.0 µg/mg) remained unsaponified in the manufactured natural soaps regardless of feedstock used. Principal component analysis (PCA) analyses showed the unsaponifiable fatty acids were different in the hibiscus bar compared to the other bars. There was a very strong correlation between the content of unsaponified C18:3n3 and C18:1n9 in all natural soaps. These results indicate that unsaponified fatty acids are important contributors to the quality and overall sensory perception and preference of natural herbal soaps following manufacturing by cold saponification. The global bar soap industry is worth approximately US$186 billion. Current trends in consumer preference have shown an increase in demand for the use of natural ingredients in personal skin care and cosmetics products. This has resulted in a steady increase in small- and medium-sized artisan hand-made or homemade soap businesses offering a variety of products with a range of natural ingredients to supply this niche market. Formulation of specialty hand-made natural soap bars by artisan soap makers involves a skillful combination of the ingredients, thoughts, and artistic creativity to produce high-quality soap bars with superior sensory characteristics that resonates with consumers ,3,4. Of The most commonly used oil sources in natural herbal soaps are mixtures of palm, coconut, olive, rice bran, and sunflower seed oils ,3. In soThree natural soaps were manufactured in this study: BB, FG, and H . VegetabNatural additives are commonly formulated into herbal soaps to modify the soap composition, quality, performance, and consumer’s sensory perception of the final product . These i2O2 equivalents/g soap). A significantly higher oxidation status was observed in FG (35.30 ± 1.34), H (37.45 ± 2.66), HA (35.49 ± 1.35), BBR (36.35 ± 1.01), and FGR . As such, the total (TOS) and lipophilic (LOS) oxidation status differentiated FG from H and BB, while the hydrophilic oxidation status (HOS) differentiated H from BB and FG. Polyphenols conferred 65–72% of the antioxidant activities observed in the natural soaps , exotic essential oils, or plant extracts (as additives) that the industry partner typically used to make their natural soaps for commercial sales. The use of these ingredients did not translate to superior consumer preference or acceptance. These findings suggest some of the more exotic additives and specialty oils used as feedstock in manufacturing natural soaps may not be producing the perceived consumer acceptance or preference, and considerations should be given to their use during manufacturing of hand-made natural soaps. This could have implications in the cost of materials used for production and potential profitability.w/v): coconut oil (30%), palm oil (20%), soybean oil (20%), olive oil (20%), castor oil (5%), and shea butter (5%). The forest grove soap was manufactured from the base bar ingredients with the following modification: soybean oil was reduced to 18%, and 2% stearic acid was added to compensate for the reduced soybean oil. The hibiscus rose hip soap was also manufactured using the base bar ingredients with the following modification: soybean and olive oils were reduced to 18% and 10%, respectively, and 2% stearic acid and 10% rice bran oils were added instead to compensate for the reduced levels of soybean and olive oils. The oils/butter mixture for each soap type were melted in a double jacketed melter at 150 °F and thoroughly mixed using an industrial immersion blender. Aliquots (8 g) of the melted oils and butter from each soap batch were obtained to determine the chemical composition of the soap feedstock prior to cold saponification. The oil samples (soap feedstock) were designated OilBB (oil from base bar), OilFG (oil from forest grove), and OilH (oil from hibiscus rose hip). Four separate batch (replicates) were independently made for each soap type.Three natural herbal soaps designated base bar (BB), forest grove (FG), and hibiscus rose hip (H) were manufactured using cold saponification. The health Canada compliant numbers for each of the natural soaps are as follows: 006324, 0.006354, and 0.006362 respectively. The base bar was designated the control bar, and contained the following oils and butter melted and mixed together . The saponification value for all oils used in the composition of the different soaps were determined using a commercial lye calculator. The cold process method herein referred to as cold saponification was used for soap production where lye (pure sodium hydroxide) was used as the base in the saponification process. The amount of sodium hydroxide required  was mixed with water and left to cool for 60 min. The cooled lye and water was added to the melted oils and butter for each soap type (see above), and the subsequent mixture was blended until thickened (about the consistency of thick cream), but before trace  was reached (the consistency of pudding). The mixture was used to fill parchment paper-lined molds, and the soaps were left to complete saponification at room temperature for 24 h. Following saponification and removal from the mold, each loaf of soap weighed approximately 1500 g, and measured 2 × 3 × 15 inches. A commercial wire loaf cutter was used to cut the loaves into 15 identical bars simultaneously, with each bar having the same shape and dimensions , and the soaps left to cure for 4 weeks. After curing, 8 g were cut with a scalpel blade from the center of each soap bar, wrapped in aluminum foil, and stored at −80 °C for chemical analyses. A total of eight batches for each soap type were made for both chemical and sensory perception analyses. Each soap type was designated as follows: base bar (BB), forest grove (FG), and hibiscus rose hip (H) based on their formulation.w/w) of rosemary extract   was added to each loaf. Both soap samples were designated BBR (base bar + rosemary extract) and FGR (forest grove + rosemary extract), respectively. To both hibiscus and forest grove, a second combination of natural additives consisting of a combination of naturally sourced colored clays (for color) and essential oils (for fragrance) were added. Specifically, 0.05% (w/w) of French green clay and essential oil blend containing patchouli and fir needles, cedar leaf, cedar wood, and cinnamon bark were added to the forest grove bar. This soap bar was designated FGA. To hibiscus, French pink clay, filtered hibiscus rose hip tea steeped in boiled water for 1–3 h , and an essential old blend containing sweet orange, pink grape fruit, bergamot, vanilla, cinnamon bark, cedar leaf, ylang ylang, cananga, lavender, and palmarosa were added as additives. This soap bar was designated HA. The amount of additives added to each soap type was determined based on the FDA approved usage of extracts/natural preservatives in cosmetics (0.02–0.05% of finished product weight). As such, 0.05% (750 mg) of the finished product weight (1500 g) was used to add the additives in this study. The additives were added to the mixture and thoroughly mixed just before molding. The same cold saponification method noted above was used to manufacture the soaps with additives. Eight grams was cut with a scalpel blade from the center of each soap bar, wrapped in aluminum foil, and stored at −80 °C for chemical analyses.To evaluate the effects of additives on the unsaponified fatty acid composition of BB, FG, and H herbal soaps following cold saponification, natural plant additives were added to each of the soaps as follows: To the base bar and forest grove soaps, 0.05% (v/v) were added. The samples were centrifuged at 10,000× g for 15 min, and the supernatant was carefully removed without disturbing the pellet. The supernatant was filtered with glass wool, and the filtrate was used without further dilution to determine the lipophilic antioxidant activity and the organic phenolic content of the soaps. The undisturbed pellets were re-suspended in 1 mL of cold 50 mM sodium phosphate buffer (pH 7.5), and centrifuged at 10,000× g for 15 min, and the supernatant was carefully removed without disturbing the pellet. The supernatant was diluted 1:10 with 50 mM sodium phosphate buffer. This diluted supernatant was used to determine the hydrophilic antioxidant activity and aqueous phenolic content.Extraction was carried out according to the methods of Thomas et al.  and Canov/v) extract was added to microplate wells to determine the hydrophilic and lipophilic phenolic content, respectively. The microplates with the mixture were incubated in the dark at room temperature for 30 min, and the absorbance was measured at 755 nm using a Synergy HT microplate reader . The results were expressed as micromole quercetin equivalents per gram of soap. Four replicates were analyzed per standard concentration or sample treatment. Values for total phenolic content were determined by summation of the aqueous (hydrophilic) and organic (lipophilic) phenolic values.The hydrophilic and lipophilic phenolic content were determined separately using a 10-fold diluted solution of Folin–Ciocalteu reagent with quercetin as a standard in the range of 0–1.0 mg/mL ,14. The 3·6H2O solution. This solution was heated to 37 °C in an oven for 15 min before use. A standard solution of 1 mM of Trolox was made using 6.25 mg Trolox in 25 mL of sodium phosphate buffer for the hydrophilic antioxidant activity or acetone:ethanol (1:1 v/v) for the lipophilic antioxidant activity. For hydrophilic and lipophilic antioxidant activities, 10 µL of sample or standard was added to the reaction medium to give a total volume of 200 µL in each microplate well. The sample mixture was incubated in the dark for 30 min and the absorbance was measured at 593 nm using a Synergy HT microplate reader . Four replicates were analyzed per standard concentration or sample treatment. The results were expressed as micromole Trolox equivalents per gram soap. Values for total antioxidant activity were determined by summation of the aqueous (hydrophilic) and organic (lipophilic) antioxidant values.The hydrophilic and lipophilic antioxidant activities were determined according to the methods of Jimenez-Alvarez et al. . This meo-dianisidine complex in the assay to produce ferric ions. The ferric ions produce a chromophore (colored complex) with xylenol orange in an acidic medium, and the amounts of oxidant molecules present in the samples can be determined via a linear relationship with known standards by measuring the absorbance at 560 nm. Briefly, a stock solution of Reagent 1 was prepared by dissolving 114 mg of xylenol orange and 8.18 g of sodium chloride in 900 mL of 25 mM H2SO4 (1.2 mL of 98% H2SO4 (d = 1.84 g/L) in 900 mL H2O). This was followed by the addition of 100 mL of glycerol to give a final composition for Reagent 1 of 150 µM xylenol orange, 140 mM NaCl, 1.35 M glycerol, and a pH of 1.75. Reagent 2 was prepared by dissolving 1.96 g of ferrous ammonium sulfate and 3.17 g of o-dianisidine dihydrochloride in 1000 mL of 25 mM H2SO4 (1.33 mL of 98% H2SO4 (d = 1.84 g/L) in 1000 mL of H2O). This reagent had a composition of 5 mM ferrous ammonium sulfate and 10 mM o-dianisidine dihydrochloride. Hydrogen peroxide in water was used as internal standards, where 10.24 µL of 30% H2O2 was added to 100 mL of deionized water to give a 1 mM stock solution. From this stock solution, serial dilutions were performed to give 0, 25, 50, 100, and 150 µM. For this assay, 35 µL of sample or standard was placed in microplate wells along with 225 µL of Reagent 1 and 10.6 µL of Reagent 2. The absorbance was then read at 560 nm using a Biotek Synergy HT microplate reader . Four replicates were analyzed per standard concentration or sample treatment, and the lipophilic and hydrophilic oxidant status was determined. The results were expressed as µM equivalents of H2O2 per gram of soap. Values for the total oxidant status were determined by summation of the aqueous (hydrophilic) and organic (lipophilic) oxidant values.This analysis was conducted using the established method of Erel . The prig for 10 min. The organic layer was retained to determine the unsaponified fatty acids in the soaps or fatty acids in the oils. The fatty acids in the mixture were methylated by taking 200 µL aliquots of the organic layer from each sample, then dried under nitrogen gas. To the dried residue, 200 µL of methanolic-HCl (3N) was added, and the samples were vigorously vortexed then incubated overnight at room temperature. Following incubation, 800 µL of deionized water was added and the samples were extracted three times using 500 µL of n-hexane each time. The hexane fractions from each sample were pooled (1500 µL), 100 µL of water scavenger (DMP) was added, and the sample was dried under nitrogen [Soap lipids were extracted using the established Bligh and Dyer method with the following modifications ,18. Brienitrogen . The metGC-MS/FID analysis was conducted on a Thermo Scientific Trace 1300 gas chromatography (GC)  coupled to a Thermo Scientific TSQ 8000 Triple Quadrupole mass spectrometer (MS)  or a flame ionization detector (FID) . Methylated fatty acids were separated with a DB23 high resolution column  using helium as the carrier gas at a flow rate of 1 mL/min. One microliter (1 µL) of each sample was applied to the injection system in split mode (20:1) using a Tri-plus auto-sampler . The oven temperature was programed as follows: the initial oven temperature of 80 °C was held for 0.5 min, then programmed to increase at 4 °C/min to 220 °C, held for 5 min at 220 °C, then increased at 4 °C/min to 240 °C, where it was held for 10 min. The methylated fatty acids were determined from the comparison of retention times and mass spectra were obtained from commercial standards  and the NIST database . The amounts of individual fatty acids identified were calculated using standard curves prepared from the standard mixtures, and values were presented as µg/mg soap .The effects of the unsaponified fatty acids on the perception of herbal soaps were conducted according to the methods of Garcia-Segovia et al.  and Sampn = 59) were separated using Fisher’s LSD test (α = 0.05) when significant differences were observed [All measurements of chemical parameters  were made in quadruplet. Analysis of variance (ANOVA) was used to determine the effects of treatments on chemical parameters. Where treatment effects were significant, the means were compared using Fisher’s LSD test with α = 0.05. For sensory perception analysis, results from the hedonic scale were analyzed using ANOVA and frequency distribution analysis, and means  appeared to be the most responsive to the variation in the choice of vegetable oils used as soap feedstock during manufacturing via cold saponification. Addition of additives in the soap formulation had a significant effect on the percent change or retention of the feedstock fatty acids as unsaponified fatty acids in the finished products. Both the antioxidants and phenolics were associated with the retention or levels of C18:1n9 and C18:3n3, but not C18:2n6 unsaturated unsaponified fatty acids in natural herbal soaps manufactured via cold saponification. These findings suggest cold saponification is an effective approach to enhance the levels of unsaponified fatty acids, phenolics, and antioxidants in hand-made natural soaps. Appealing smell was the greatest influencer of consumer preference, while color and appealing smell were the best indicators of natural soaps’ estimated pricing and consumers’ perceived quality, and consequently acceptability. These findings are of major significance to artisanal production of natural herbal soaps because antioxidants, polyphenols, and unsaponified unsaturated fatty acids appear to be major determinants of natural soap quality, consumer perception, and preference of the final products. One of the most significant findings in this study was that the base bar used as control in this study had the best overall preference compared to the other soaps formulated with specialty or exotic plant oils as feedstock and additives. These findings suggest some of the more exotic additives and specialty oils used to manufacture hand-made natural soaps may not be producing the perceived consumer acceptance or preference, and that artisan natural soap makers should give careful considerations to their use during the manufacturing of hand-made natural soaps. This work provides some baseline information regarding natural hand-made soaps manufacturing, which is very sparse in the scientific literature. The hope is that the information presented will stimulate additional studies by other researchers in the scientific community, to further improve the knowledge that may be of value to the growing specialty hand-made soap industry.


In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis. mTORC2 activates Akt, a regulator of cell growth and metabolism, however, the role of mTORC2 in adipocytes is incompletely understood. Here the authors report that a mTORC2-Akt axis specifically activates ACLY to promote lipid synthesis and histone acetylation during brown adipocyte differentiation. Amino acids and insulin stimulate mTORC1 to promote protein, lipid, and nucleic acid synthesis through well-defined downstream signaling networks. mTORC2 promotes glycolysis and lipid synthesis; however, its regulation and downstream mechanisms are less well defined.A cell’s capacity to sense and respond to nutrients is essential for metabolic homeostasis and losing this flexibility is a hallmark of diabetes and cancer. The mammalian target of rapamycin (mTOR) kinase is a master regulator of intracellular metabolism that responds to insulin signaling and nutrient availability. Its biochemical functions are split between two complexes called mTOR complex 1 (mTORC1), which contains the essential subunit Raptor, and mTOR complex 2 (mTORC2), which contains the essential subunits Rictor and Sin13. HM phosphorylation cooperates with 3-phosphoinositide-dependent kinase 1 (PDK1)-dependent phosphorylation in the T-loop  to maximally stimulate AKT catalytic activity5. Growth factors facilitate AKT phosphorylation by stimulating AKT and PDK1 recruitment to the plasma membrane through pleckstrin homology (PH) domains, which bind phosphatidylinositol 3,4,5-triphosphate (PIP3). mTORC2 and PDK1 additionally co-regulate the related AGC family kinases SGK and PKCα9. These substrates lack PH domains, thus their interaction with mTORC2 and PDK1 is likely facilitated by a different mechanism4. However, the temporal and spatial regulation of mTORC2 signaling remains poorly understood.The best understood biochemical function of mTORC2 is to phosphorylate the hydrophobic motif (HM) site in AKT 5; however, its exact function in downstream AKT signaling in vivo remains elusive because, in many tissue-specific conditional Rictor knockout (KO) models, mTORC2 loss has seemingly minimal-to-no effect on the phosphorylation of several AKT substrates10. For example, conditionally deleting Rictor in brown adipose tissue (BAT)  or in white adipose tissue (WAT) (with adiponectin-cre) ablates AKT HM phosphorylation without obviously impairing AKT activity toward classic substrates, including PRAS40, TSC2, GSK3β, AS160, and FoxO114. Yet, despite seemingly normal AKT signaling, Rictor loss profoundly downregulates transcription of ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), which catalyze de novo lipogenesis (DNL)12. Rictor loss also reduces glucose uptake and inhibits the expression of ChREBPβ13, which is a constitutively active isoform of the carbohydrate response element binding protein (ChREBP) transcription factor that stimulates carbohydrate and lipid metabolic gene expression15. Similarly, inducible deletion of Rictor in brown preadipocytes has seemingly no effect on downstream AKT signaling, yet renders these cells incapable of differentiating in vitro11. Nevertheless, expressing recombinant AKT1 containing a phospho-mimetic S473 residue in Rictor-deficient preadipocytes rescues differentiation. The latter observation suggests that mTORC2 may be more essential for some AKT actions that are required for brown adipocyte differentiation and de novo lipogenesis, but dispensable for many others.Many models show mTORC2 as an obligatory AKT activator16, PKA18, mTOR19, or the branched chain ketoacid dehydrogenase kinase20 can reportedly phosphorylate this site. Nevertheless, because ACLY functions at the interface of glucose-dependent DNL and epigenetic control of gene expression, it is poised to be a key link between hormonal signaling and carbohydrate and lipid metabolism in adipocytes.ACLY cleaves extra-mitochondrial citrate to generate acetyl-CoA, which is the precursor for glucose-dependent de novo lipid and cholesterol biosynthesis. Acetyl-CoA is also used to acetylate lysine residues on histones and metabolic proteins to regulate gene expression and enzyme activity, respectively. ACLY serine 455 lies within a basophilic phosphorylation motif (RxxS) that is similar to the AKT consensus motif (RxRxxS/T). Phosphorylation of this site stimulates ACLY activity; however, the serine 455 kinase remains controversial as AKTRictor-deleted brown adipocytes and reveal mTORC2’s central role in controlling nuclear–cytoplasmic acetyl-CoA synthesis.Here we test the hypothesis that mTORC2 promotes brown adipocyte differentiation and glucose-driven DNL (gluco-lipogenesis) through a distinctly mTORC2-dependent AKT pathway. To do this, we analyzed mTORC2-regulated AKT phosphosites and metabolites by mass spectrometry. This led us to identify a subset of AKT substrates and metabolites that are regulated by mTORC2, including the enzyme ACLY and its product acetyl-CoA. We provide evidence that ACLY functions downstream of a uniquely mTORC2-dependent AKT pathway required for DNL, differentiation, histone acetylation, and ChREBPβ and gluco-lipogenic gene expression. Substrate utilization studies suggest an additional role for mTORC2 in promoting acetyl-CoA synthesis from acetate through acyl-CoA synthetase short chain family member 2 (ACSS2). These data uncover a previously unappreciated selectivity in mTORC2-dependent AKT signaling in Rictor-iKO precursor brown adipocytes (Rictor-iKOPBAs) and isogenic control brown preadipocytes. We designed the assay to quantify phosphopeptides encompassing regulatory phosphorylation sites on AKT, previously reported AKT and SGK substrate sites, and other potential mTORC2 effectors. Rictor-iKOPBAs maintain growth-factor-stimulated AKTT308 phosphorylation, albeit at lower levels, and seemingly normal phosphorylation of AKT substrates -glucose (10 mM) and physiological levels of unlabeled acetate (100 μM) or physiological levels of -acetate (100 μM) and unlabeled glucose (10 mM) for 1 h prior to measuring acetyl-CoA. Compared to their isogenic controls, the percentage of labeling of acetyl-CoA from both glucose and acetate is unchanged in Rictor-iKOPBAs -glucose (NEC042V250UC) was purchased from Perkin Elmer. MK2206 (S1078) was purchased from Selleck Chemicals. T3 (T2877), dexamethasone (D1756), IBMX (I5879), and insulin (I2643) were from Sigma-Aldrich. [U-13C] glucose (CLM-1396-1) and  acetate (CLM-440-PK) were purchased from Cambridge Isotope Laboratories, Inc.GLUT-1 (ab652) and H3K27Ac (ab4729) were purchased from Abcam. HK (A0994) and horseradish peroxidase (HRP)-goat anti-Rabbit were purchased from ABclonal. HRP-conjugate anti-mouse (w402b) was purchased from Promega. H2BK (07-373), H4K (06-866), and H3K (06-599) were purchased from Millipore. Flag (F7425) was purchased from Sigma-Aldrich. All other antibodies including Rictor (2140), PRAS40 (2691), AKT (9272), GSK3b (9315), ACC (3676), ACLY (4332), FASN (3180), PPARg (2443), ACSS2 (3658), S6 (2217), S473-AKT (4058), T308-AKT (4056), S455-ACLY (4331), T246-pPRAS40 (2997), HA-tag (2367), S9-GSK3b (9315), S235/236-S6 (4858), H3 (9715), H3K9Ac (9649), H3K14Ac (7627), and a-Tubulin (2125) were purchased from Cell Signaling Technologies. 4-OHT was obtained from Toronto Research Chemicals. D- was then added, and incubation was continued for an additional 10 min. The medium was then removed and cells were washed three times with KRH medium to terminate the assay. Cells were then lysed in 1% triton, mixed with scintillation buffer, and the uptake of 3H glucose was quantified in counts per minute (cpm) using a scintillation counter. The cpm values were normalized to the protein concentration of each sample.Cells were preincubated for 3 h in a medium without glucose (KRH medium (Gibco plus 0.5% BSA + 2 mM pyruvate). Deoxy-Rictor-iKO cells were serum deprived for 12 h (unstimulated samples) and then some were treated with 150 nM insulin for 15 min (stimulated). All cells were washed 3 times with cold PBS, snap-frozen in liquid nitrogen, and thawed in a lysis buffer ). Each well of a 6-well plate was scraped in 125 μL of ice-cold lysis buffer, and 2 wells were pooled for each replicate. Lysates were probe-sonicated two times, and clarified by centrifugation at 16,000 × g. Protein content was measured by a BCA assay (Pierce). Samples were reduced for 45 min at 55 °C using 5 mM dithiothreitol, alkylated with 15 mM iodoacetamide for 30 min in the dark at room temperature, and quenched with an additional 5 mM of dithiothreitol at room temperature for 15 min. Urea was diluted to 1.5 M using 50 mM pH 8.2 Tris, and protein was digested for 16 h using 5 μg/mL trypsin at 37 °C. Digested peptides were de-salted using 50 mg Sep-Pak cartridges (Waters) and dried by vacuum centrifugation. Phosphopeptides were enriched using PureCube Fe-NTA magnetic beads (Cube Biotech) in a KingFisher magnetic bead processor (Thermo). Dried phosphopeptide samples were prepared for analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) by dissolution in 4% formic acid and 3% acetonitrile.Control and 52, we referenced an in-house database of mouse phosphoproteome data gathered from several different tissues using data-dependent acquisition (DDA) to determine the best phosphopeptide(s) with which to represent each phosphorylation site. Specifically, for each phosphorylation site, we determined which charge state and peptide cleavage form was the most frequently observed in the DDA data. Importantly, this database also enabled accurate prediction of the chromatographic retention time. In some cases, phosphorylation sites were represented by multiple peptides in the preliminary target list because it was not obvious which peptide form to choose. To refine the preliminary target list, three separate PRM assays were used to measure these phosphopeptides in insulin-stimulated WT cells with wide (+/−2.5 min) windows for retention time prediction. Peptides that were not detected or contained complete overlap of phosphorylation sites with another detected peptide were removed from the list, and all retention time predictions were re-calibrated. This final target list was then used for PRM analysis of all the samples with narrow (+/−1.5 min) windows for retention time prediction.To screen for mTORC2-dependent phosphorylation, we designed a targeted, parallel reaction monitoring (PRM) mass spectrometry method that was informed by manual literature curation and prior preliminary experiments. To design the list, we curated a preliminary list of 228 phosphopeptides representing previously characterized AKT substrates, regulatory phosphorylation sites on AGC kinases, phosphorylation sites on proteins with known regulatory function in insulin signaling and/or brown fat, and phosphorylation sites with potential mTORC2 dependence according to preliminary, unpublished data. According to a previously reported methodm/z at 70,000 resolution with an AGC target of 3 × 106 and a maximum injection time of 240 ms. The top 20 most abundant precursors per MS scan were isolated with a 2-m/z window, an AGC target of 2 × 105, a maximum injection time of 120 ms, and 40 s dynamic exclusion. Isolated precursors were fragmented by high-energy collision-induced dissociation at a normalized collision energy of 27%, and MS/MS were acquired at 17,500 resolution. Targeted LC-MS/MS using PRM was performed on the same instrument set-up as for DDA. The instrument methods were mostly the same as for the DDA analysis, except that MS/MS spectra were acquired according to the predefined schedule of precursors representing the phosphopeptide target list, as described above. Every 20 scans, a full MS scan was acquired as described above for DDA. All MS and MS/MS spectra for DDA and PRM experiments were collected in centroid mode. PRM data were analyzed using Skyline53, and all chromatographic peaks and phosphorylation site localizations were manually inspected. For additional verification of phosphorylation site localization, PRM data were searched using Comet (v2015.02.5)54 and filtered at 1% FDR using Percolator55. Each MS/MS spectrum was scored for localization using Ascore56. Ascore values representing confidence of site localization were plotted vs retention time and manually compared to the fragment chromatographic elution peaks in Skyline. Phosphorylation sites were stratified into three categories of mTORC2 sensitivity by calculating ANOVA statistics to determine the significant effects of Rictor loss and correcting to q-values using the Benjamini–Hochberg method. Class I sites had q ≤ 0.05 and at least a 2-fold difference between control and Rictor-iKOPBA. Class III sites had q ≤ 0.05 and at least a 1.33-fold difference between control and Rictor-iKOPBA. All the phosphoproteomic data generated during this study are available and were deposited in Massive.Ucsd.edu [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=2335fcac3c184be69e7efe068387f771].Targeted phosphoproteomic analysis of preadipocyte phosphorylation sites was performed using LC-MS/MS. To normalize for injection amount during LC-MS/MS analysis, we measured all samples using DDA and identified 12 phosphopeptides that correlated strongly with the total MS signal and spanned most of the retention time space. DDA analysis was performed on a Thermo QExactive mass spectrometer connected to a Thermo Easy-nLC II. Chromatographic separation was performed on a 30-cm length column with a 100-μm internal diameter, packed with Reprosil 1.9 μm C18 beads (Dr. Maisch GmbH). The LC method consisted of a 49-min linear gradient from 9% to 25% acetonitrile in 0.15% formic acid. Data were acquired throughout a 70-min total time. Full MS scans were acquired from 300 to 1500 Cells were incubated in serum-free media for 12 h and then incubated for 3 h with fresh DMEM medium. Samples were prepared by performing an 80% methanol extraction and stored at −80 °C until shipping. Polar metabolite profiling methods were developed at the Whitehead Institute for Biomedical Research Metabolomics core facility, where the measurements were performed. Reference standards of each metabolite were used to determine chromatographic retention times and MS multiple reaction monitoring transitions, declustering potentials, and collision energies. All metabolites were normalized to the protein concentration of each sample.TM UCD200 sonicator . Size of fragments obtained (between 200 and 1200 bp) was confirmed by electrophoresis. Soluble chromatin was collected after centrifugation at 16,000 × g at 4 °C for 10 min. Soluble chromatin (1%) was kept as input control. Soluble chromatin was precleared with 2.5 μg/mL of protein-A-Sepharose at 50% at 4 °C in rotation for 1 h and beads were spin down at 5900 × g for 3 min at 4 °C. After centrifugation, supernatants were collected and specific antibodies  were added. Mixtures were incubated overnight at 4 °C for 8 h in rotation and then incubated for 2 h at 4 °C in rotation with protein-A-Sepharose at 50% (Roche). Beads were collected and washed sequentially at 4 °C for 10 min with wash buffer A ), wash buffer B ), LiCl buffer (20 mM Tris-HCl (pH 8), 1 mM EDTA, 250 mM LiCl, 0.5% deoxycholate, and 0.5% NP-40), and TE buffer (10 mM Tris-HCL (pH*), 1 mM EDTA). Immunoprecipitates were eluted two times with elution buffer elution buffer (50 mM Tris-HCl (pH 8), 1 mM EDTA, and 1% SDS). Reversion of cross-linking was performed overnight by heating samples and input controls at 65 °C. Samples were then treated with 0.2 mg/mL RNAse A (Qiagen) and incubated for 30 min at 37 °C followed by addition of 1 μL of (20 mg/mL) Proteinase K (Promega), and samples were incubated at 45 °C for 1 h. DNA was then purified using the QIAquick Spin Kit (Qiagen). Real-time reverse transcriptase PCR was performed with primers listed in Supplementary Table Cells were cross-linked with 1% paraformaldehyde for 15 min at room temperature. The reaction was quenched for 5 min at room temperature by adding 0.125 M glycine. After three washes with 1× PBS, cells were lysed with lysis buffer  supplemented with protease and deacetylase (TSA) inhibitors. Lysates were sonicated on ice using a Diagenode Bioruptor3C6-glucose and 100 μM normal or 13C2 acetate at 37 °C for 1 h. Samples were harvested in cold PBS, centrifuged 1000 × g, 5 min, and the pellet was quenched in 10% (w/v) ice cold trichloroacetic acid in water, and then frozen at −80 °C until extraction and analysis on a QExactive Plus (Thermo) mass spectrometer coupled to an Ultimate 3000 high-performance LC. For quantitation, the abundance of acyl-CoA species was determined by interpolation to a linear standard curve. For isotopic tracer analysis, isotopic enrichment was calculated to compensate for the non-linearity of isotopic enrichment using the FluxFix calculator57.Cells were fasted for 12 h and then incubated in DMEM without glucose plus 5 mM of normal or 14C]-glucose. Chloroform extraction was performed, and labeled lipids were measured using a scintillation counter. Each sample was normalized to total protein concentration58.Cells were incubated for 3 days with DMEM in which 0.01% of the total glucose concentration of the medium was comprised of D-[U-http://imagej.net/Welcome) and in each case shows the average quantification of four images representing each quadrant of the well.Cells were fixed with 4% paraformaldehyde, washed with distilled water, and treated with 100% propylene glycol twice for 5 min followed by a 15-min incubation with Oil Red O solution. Then cells were washed with 85% propylene glycol for 3 min and rinsed twice in distilled water. Oil Red O stain was quantified with the ImageJ software (Cells or tissues were lysed with Qiazol (Invitrogen) and total RNA was isolated with the RNeasy Kit (Invitrogen). Equal amounts of RNA (2 μg) were retro-transcribed to cDNA using a high-capacity cDNA Reverse Transcription Kit . Quantitative real-time PCR was performed in 10 μL reactions using a StepOnePlus real-time PCR machine from Applied Biosystems using SYBR Green PCR master mix  according to the manufacturer’s instructions. Relative mRNA expression was determined by the ΔΔCt method and Tbp expression was used as a normalization gene in all conventional PCR with reverse transcription experiments. Primer information is listed in Supplementary Table p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, a represents ***p < 0.001 vs control (EV), and b represents ***p < 0.001 vs iKO (EV) unless stated otherwise. Student’s t test or two-way ANOVA with the Sidak’s test, as appropriate, was used to determine statistical significance. Statistical analysis was done using GraphPad Prism.Data are presented as mean + SEM; *Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Reporting Summary


Ginkgo biloba seeds protein isolate (GPI). The Ginkgo protein hydrolyzates (GPHs) with the maximum degree of hydrolysis (DH) and ACE inhibitory activity were selected, and ultra-filtered to obtain components with different molecular weights (MW) . The components with MW of <1 kDa showed better ACE inhibition (IC50:0.2227 mg/mL). Purification and identification by Sephadex G-15 gel chromatography and LC-MS/MS conferred three new potential ACE inhibitory peptides . Molecular docking depicting the inhibitory mechanism for ACE inhibitory peptides indicated that the peptides bound well to ACE and interacted with amino acid residues at the ACE active site.Alcalase, dispase, trypsin, and flavourzyme were used to hydrolyze the extracted  Biologically active peptides are the general term for different peptides which constitute different compositions and arrangements of natural amino acids. They are known to possess a variety of biological functions such as antioxidant, immune-promoting, hormone-modulating, antibacterial, antithrombotic, antiviral, and antihypertensive activities due to multifunctional compounds derived from animal or plant proteins . Moreovein vitro hypotensive activity of peptides has been primarily determined by measuring the inhibitory activity of angiotensin converting enzyme (ACE) which is a dipeptidyl carboxypeptidase located on the cell membrane and can cause high damage by disrupting the balance between the two hormonal systems which regulate blood pressure and fluid balance , Hippuric acid were purchased from Sigma  and Yuanye Bio-Technology , respectively. Captopril was procured from MedChem Express .The G. biloba L. seeds were dried at 45°C, then crushed into powder and sieved through 80 mesh. The powder was freeze-dried after dephenolation and degreasing treatment. The resulting G. biloba seeds powder was dissolved in DW . The above suspension was stirred and extracted for 12 h followed by centrifugation at 10,000 g for 30 min. The obtained supernatant was set to the isoelectric point of ginkgo protein pH 4.62 and incubated for 60 min followed by further centrifugation at 10,000 g for 30 min. The obtained precipitate was resuspended using distilled water, and the solution was adjusted to pH 7. Subsequently, the obtained GPI were freeze-dried until next use.The g for 30 min to obtain the individual supernatants obtained from four enzymes.For this, 4% GPI solution was prepared and hydrolyzed separately with four proteases at their optimal hydrolysis conditions for 5 h. The enzyme dosages were 2,000 U. After hydrolysis, enzymes were inactivated at 90°C for 10 min and the solution was centrifuged at 3,000 The degree of hydrolysis (DH) was calculated by referring to the method of b was the molar concentration of NaOH solution; MP was the mass (g) of protein; α designated the average degree of dissociation in protein substrates during hydrolysis; htot was the total number of peptide bonds in the protein.where, B represented the amount (mL) of NaOH added to keep the pH constant during the hydrolysis process; NSample ultrafiltration was carried out by using MSC300 cup type ultrafilter . The sample was added from the feed port and the nitrogen bottle was connected to the hose fitting of the ultrafiltration cup. Then after, the ultrafiltered cup was placed and packed on the magnetic stirrer connected with the nitrogen gas bottle with a pressure not more than 0.22 MPa. Samples were sequentially passed through 1, 3, 5, and 10 kDa ultrafiltration membranes and four components were obtained . The four fractions were collected, freeze-dried, and then used to test the ACE inhibitory activity. The components with higher ACE activity were selected for further purification as per the procedure given by 2[B4O5(OH)4] (pH 8.3) with 0.3 M sodium chloride. Then after, 50 μL of sample and 150 μL of the substrate were added to a centrifuge tube, mixed and allowed to stand in water at 37°C for 5 min. Subsequently, the ACE solution (50 mU/mL) was added and incubated at 37°C for 45 min. The reaction was terminated by adding 250 μL of 1M HCl and the solution was filtered through 0.45 μM nylon syringe filter before being analyzed by RP-HPLC  on an Inertsil ODS-3 . The mobile phase comprised of distilled water: acetonitrile = 75:25  with a flow rate of 0.5 mL/min and detection at 228 nm. Captopril was used as a control and the inhibitory activity (%) was determined as below:This test was performed as per the descriptions are given by the previous report by where A was the peak area of hippuric acid with the sample, B was without the sample.The purified samples were analyzed by Q Exactive  using an Easy-nLC 1000 . The sample was desalted and lyophilized, reconstituted in 0.1% FA solution and stored at −20°C until next use. The two mobile phases were as follow: mobile phase A constituted an aqueous solution of 0.1% formic acid, and mobile phase B was a 0.1% formic acid aqueous solution of acetonitrile (acetonitrile was 84%). After equilibration of the column with 95% of the A solution, the sample was loaded from the autosampler to the Trap column. The mass-to-charge ratio of fragments of peptides was collected as follow: total 20 fragment patterns were acquired after each full scan. The mass spectrometry test raw file was searched using Mascot 2.2 software for the corresponding database.Potential ACE inhibitor peptides identified by LC-MS/MS were synthesized at GL Biochem . The purity of the synthetic peptides was further confirmed by HPLC which was greater than 95%.G. biloba peptide was tested using the method of The kinetic inhibition model of 1) was downloaded. The three-dimensional structure of G. biloba peptides was drawn using the molecular simulation software Discovery Studio 3.5. The hydro processing was performed and the energy was minimized by the CHARMM program. In addition, Zn2+ was retained in the ACE model. The DS 3.5 software scored the docking result according to its own scoring function, based on the scores and the combined free energy of each result. Among all the results, a better matching result was selected.For this, three-dimensional structure file of ACE protein (PDB ID: 1O8A) from RCSB Protein Data Bank (PDBP ≤ 0.05).One-way ANOVA, followed by Duncan’s multiple-range tests using SPSS Statistics 20.0  was used for data analysis with a significance difference , 44.04% (IC50 = 1.765 mg/mL), respectively. Subsequently, activity gradually increased with the lower MW and reached the highest level  at <1 kDa.The ACE inhibitory activity of the GPHs and the resulting fractions was significantly dependent on MW as shown in Figures Alcalase is often used for the preparation of active peptides due to its broad specificity and strong ability to degrade proteins. From the ultrafiltration treatment, it was revealed that the ACE inhibitory activity improved with the decreasing MW of the polypeptide. Therefore, the peptide of the <1 kDa fraction was further purified using Sephadex G-15 column. It can be seen from Figure 50 values of TNLDWY, RADFY, and RVFDGAV were 1.932, 1.35, and 1.006 mM, respectively  was separated by gel filtration chromatography, and the amino acid sequence was identified using LC-MS/MS. Thus, three new ACE inhibitory peptides were obtained: TNLDWY (1.932 mM), RADFY (1.35 mM), and RVFDGAV (1.006 mM) which showed strong ACE inhibitory activity. According to report by In order to further purify the highly active ACE inhibitory peptide, the Qula casein exhibited a competitive inhibition mode. However, for non-competitive inhibitors, they bind to sites other than the substrate binding site of the enzyme and ultimately affect the binding of the substrate to the enzyme. Similar patterns were observed in case of food-borne inhibitory peptides such as PFPGPIPN from Qula casein, and the hazelnut peptide YLVR , most of them were competitive inhibitors and they attached to the ACE enzyme in order to prevent the binding of the substrate HHL. A similar study by ide YLVR . In thiside YLVR .2+-dependent carboxydipeptide enzyme, in which Zn2+ is an important part of the ACE active center . S1 ; S2 ; and S1′ (Glu162 residue). At the active site of ACE, two histidines (His383 and His387) together with Glu 411 constituted a Zn2+ ligand. It has been reported that the inhibition of peptide-induced ACE activity may be achieved by the combination of hydrogen bond-stable enzyme-peptide complexes  were obtained from the peptide fraction (<1 kDa) by LC-MS/MS. RVFDGAV showed the highest ACE inhibitory activity with an IC50 value of 1.006 mM and appeared as a competitive inhibitor. Molecular docking results indicated that the peptides could firmly bind to ACE and further interacted with amino acid residues at the ACE active site. Our results indicated that the peptides obtained by enzymatic hydrolysis of alcalase exhibited effective ACE inhibitory activity in vitro, which make them potential candidates for the development of functional foods or anti-hypertensive drugs in the future.In this study, F-FM, HW, C-KW, and KT were involved in the project design, carried out most of the experiments, and drafted the manuscript. Z-JW and LJ contributed to the experimental design, manuscript preparation, and submission. All authors participated in writing the manuscript and/or revised it critically for important intellectual content.HW was employed by Anhui Habopharmqnceutical Co., Ltd. Z-JW was employed by Anhui Qiangwang Seasoning Food Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aims to investigate screw insertion sites on the pubic body and the safe screw insertion parameters of a plate-screw fixation system based on the premise of avoiding damage to the inguinal canal and disruption of the rectus abdominis at the pubic symphysis and pubic crest. Excluding cases with poor image quality, tumors, malformations, and fractures, the data of 80 healthy adults  undergoing a computed tomography (CT) scan of the pelvis between January and June of 2017 were collected from Shandong Provincial Hospital. The CT scans were imported to Mimics® software to reconstruct three-dimensional pelvic models. A 3.5 mm pelvic reconstruction plate was placed starting at the outer edge of the pubic tubercle and along the pelvic brim. The two innermost screw insertion sites were marked. The safe range for the screw insertion sites was then determined. The screw insertion plane was selected to measure the safe screw insertion parameters. The length of the screw, the direction of insertion, and intersex differences were then explored via statistical analyses. The medial inclination angles (MIAs) of the screw insertion plane for males and females were 30.42±7.95° and 32.88±10.65°, respectively, with no statistically significant differences. For the medial screw, the maximum anterior inclination angle (MAIA), the maximum screw length, and the maximum posterior inclination angle (MPIA) were 46.51±4.01°, 12.40±9.53 mm, and 11.78±10.22° on average, respectively, with no significant differences by gender (P>0.05). For the lateral screw, the MAIA was 10.35±9.46° and showed no gender differences (P>0.05), but the male group had a greater MPIA  and maximum screw length . For the tangential screw, the maximum screw length, MIA, and anterior/posterior inclination angle (APIA) were 52.19±8.33 mm, 31.65±9.42°, and 7.53±10.18°, respectively, with no significant differences in the angles by gender (both P>0.05), although the screw length in the male group was significantly longer than that in the female group. Insertion of two screws into the pubic body through a plate from the lateral side of the pubic tubercle is safe and can maintain the origin of the rectus abdominis and the integrity of the inguinal canal compared to traditional plate-screw fixation. Considering that the pubic body is thinner on the lateral side, we suggest a more medial inclination angle for the lateral screw. The pelvis is a complicated structure comprising the posterior sacrum and bilateral hip bones and exhibits considerable variations in geometrical and mechanical features. Tile et al.  found thCurrently, various fixation methods are available for unstable pelvic outcomes with anterior ring fractures –13. The Pelvic X-ray CT data of patients admitted to our hospital for reasons other than fracture between January and June of 2017 were selected. The pelvic structures of the included participants were normal. The 80 adult cases identified included 40 males and 40 females with an average age of 51.65±12.46 years and an age range of 21 to 83 years. None of the patients had anatomical abnormalities or diseases such as pelvic tumors, fracture, malformation, or severe osteoporosis. The included patients had normal pelvic structures and good bone density. The pelvic CT data were saved in digital image and communications in medicine (DICOM) format. The CT scanning parameters were as follows: a 512×512 matrix, 100-140 kV voltage, 360 mA current, 1-mm interslice interval, and 1-1.5 mm slice thickness . The 3D reconstructions using the CT data, screw insertion simulation, and parameter measurements were performed using Mimics 19.0 software . The hospital ethics committee approved this study under ethical clearance number 2018-102.The pelvic CT data in DICOM format were imported into Mimics 19.0. Hemipelvis masks were rapidly obtained through CT bone segmentation. These masks were subjected to 3D reconstruction after smoothing.The commonly used 3.5 mm low-profile reconstruction plate (provided by DePuy Synthes) was measured. The width was 10 mm, the interval between the holes was 13 mm, and the first hole was 6.5 mm from the tip of the plate . The plaA 3.5 mm cylinder was employed to simulate the pathway of the screw from the outer screw insertion site, L. The screw was inserted tangential to the inner cortex of the bone to ensure that it remained within the bone. This screw was referred to as the screw at the line tangential to the obturator. When observing from the outside, the screw insertion region was located at the inner pubis , left. WCross-section 1 was determined using the perpendicular line from point L to the pelvic boundary and the axis of the screw at the line tangential to the obturator. Cross-section 2 was defined by the plane orthogonal to Cross-section 1 and along the axis of the screw at the line tangential to the obturator . Cross-s1, at the maximum anterior inclination position of the inner screw were measured. Similarly, the MAIA, MIPA, and length of the outer screw, L2, at the maximum anterior inclination position were measured . The angle between the screw and the reference line when maximally leaning backward was the maximum posterior inclination angle (MPIA) , left. Ameasured . The MIA Figures . BecauseThree experimenters who were familiar with Mimics software performed the measurements following the aforementioned steps. The averages of the measured parameters were then subjected to statistical analysis.±s) and analyzed using SPSS 19.0. A one-sample Kolmogorov-Smirnov test was employed to ensure that the quantitative data were normally distributed and to examine the homogeneity of variance. Intersex comparisons of the measurement data were performed using two independent-samples t-tests. Differences with P<0.05 were considered significant.All measurement data were expressed as the mean±standard deviation . The MAIA of the inner screw was 12.40±9.53°, L1 was 46.51±4.01 mm, and the MPIA was -11.78±10.22° (note that the minus sign indicates posterior inclination both here and in the following sections), with no significant differences in these three parameters between males and females ( P<0.001). No differences in the APIA or MIA were found between the sexes, with values of 7.53±10.18° and 31.65±9.42°, respectively. ( P<0.05). However, the vertical distance between the pubic tubercles was shorter for females than that for males .The MIAs of the inner screw insertion planes were 21.21±4.05° for males and 25.86±8.54° for females, and the intersex difference was significant  was fixed. A reduction in the inward inclination or an adjustment of the anterior/posterior inclination may result in bone matter penetration and cause potential damage. In clinical practice, one should avoid screw insertion at this location.The average MIA of the inner screw insertion plane was 21.21±4.05° for males and 25.86±8.54° for females. Under the same conditions and with the screw insertion method employed in our study, the distance between the upper and lower boundaries of the pubic symphysis was shorter, and the inner inclination angle was greater when the screw insertion site was more outward . The measurement results indicated that the distance between the pubic tubercles was greater for females than that for males, but the distance between the upper and lower boundaries of the pubic symphysis was shorter for females than that for males. Therefore, the greater MIA at the screw insertion plane for females may be due to the greater distance between the pubic tubercles and the shorter vertical distance of the pubic symphysis. No significant intersex differences were found for the MAIA, MPIA, or LTraditional anatomical studies using cadavers are destructive. When multiplane and cross-cutting are required, bones must be dissected into pieces. The operation is difficult and not conducive to research. Errors may result from human factors and affect the accuracy and reliability of study results. In addition, measurement data may be irreproducible. The combination of digital CT data and Mimics software can substantially reduce the resources needed for research, and the corresponding results are reproducible and can be validated with a high level of accuracy. These results can also guide clinical practice , 21. ThiThe greatest challenge in digital orthopedics research is image segmentation. Post-image segmentation with 3D reconstruction is the foundation of future research. The precision of image segmentation directly affects the results of follow-up studies and therefore affects clinical practice. This study regarding bone structures was based on CT data. The principle of image segmentation is based on CT values. The higher density of bone provides greater contrast on CT images compared with nearby soft tissues, allowing better differentiation between structures. Therefore, with better image segmentation precision, the reconstructed 3D bone structure is closer to the actual structure. Importantly, however, segmentation of bone versus soft tissue may be worse in cases with severe osteoporosis, and the research results may be adversely affected. With upgraded software, automation, intelligentization, and multifunctional operations can be further advanced. Accordingly, segmentation, reconstruction, meshing, and presurgical design become more realistic, convenient, and efficient.The measurements in this study have not been performed before, although similar surgical methods are available. Traditionally, the starting point of the rectus abdominis should be dissected and the inguinal canal should be exposed to protect the spermatic cord. What we are exploring is not cutting off the origin of the rectus abdominis. Meanwhile two screws can still be inserted into the pubic body. Our study found that, by placing the plate in this way, the two screws on the medial side of the plate can penetrate into the pubic body, which has a large safety space. We measured the parameters of the safety space and the length of screws, compared the differences between men and women, proved the feasibility of the surgical method, and provided parameters. It provides theoretical support for clinical application.The shortcoming of this study is the small sample size. More subjects meeting the necessary criteria should be included in our next phase of research. The accuracy and consistency of the measurement data can be further validated using cadavers.The insertion of two screws into the pubic body through a plate from the lateral side of the pubic tubercle is safe and can maintain the origin of the rectus abdominis and the integrity of the inguinal canal compared to traditional plate-screw fixation. Considering that the pubic body is thinner on the lateral side, we suggest a more medial inclination angle for the lateral screw. On one hand, penetration into the obturator can be difficult and may result in damage to the obturator nerve and blood vessels. On the other hand, greater screw introversion corresponds to a wider anteroposterior range. We have proved the feasibility of this fixation in this study. Our further study is to study the difference in fixed strength with traditional operation  through biomechanical study.


Infectious bronchitis is an economically important disease with worldwide distribution. Information is available on the presence of infectious bronchitis virus in commercial chicken in parts of Ghana but there is no information on free-range poultry and guinea fowls in the country. Possible IBV infections among free-range chickens and guinea fowls in Abokobi and Frafraha communities in the Ga-East district of the Greater Accra Region of Ghana were investigated using serology and PCR. Blood, tracheal, and cloacal swabs were obtained from 219 free-range chickens and guinea fowls with no respiratory symptoms and no history of IBV vaccination. Sera were evaluated for IBV antibodies by ELISA using commercial IBV test kit from IDEXX, Inc., USA. Swab samples were evaluated for S1 glycoprotein gene by one-step RT PCR. All the swab samples tested negative for IBV. 16% of all tested sera were positive for IBV. IBV seroprevalence in guinea fowls was 0%. 21.2% of sera from local chickens were positive for IBV. The seroprevalence of IBV among local chickens from Frafraha was 30% and that of Abokobi was 7.7%. This study shows exposure of local chickens in the study communities to IBV. Poultry production, including chickens, ducks, and guinea fowls, is an ancient agricultural activity worldwide  . In GhanThe free-range guinea fowl and local chicken are raised with minimum capital input from the farmer. They scavenge for food themselves and are occasionally supplemented with cereals such as maize and millet and kitchen wastes from the household –10. TheyThese local birds, with the limited care, are believed to be generally hardy and resistant to various poultry diseases . In spitNewcastle disease is considered an important disease of local chickens in many African countries and is often diagnosed by experience, with little or no laboratory investigations , 9, 14 . The respiratory form of the disease is characterized by tracheal rales, cough, sneeze, and nasal discharge and gasping. Infections of the oviduct and kidneys cause egg production losses, poor egg quality, urate deposits in kidneys, and increased mortality   types of samples were taken. Blood was drawn from the neck vein of the bird with a disposable 2 mL syringe and needle and transferred into a 4-mL plain vacutainer tube. Tubes were prelabelled and slanted on racks at an angle of about 45°C to facilitate clotting and sera separation. Using rayon swabs , the trachea and cloaca of each bird were then swabbed separately and placed into a labelled microcentrifuge tube containing 1 mL viral transport medium (VTM). Tubes were immediately placed on ice in a cool box. Basic data of flock size, age of birds, and reasons for keeping the animals were collected from the owners by questionnaire administration. Samples were transported to the CSIR-Animal Research Institute, Frafraha, within 3 hours of collection.In the laboratory, swabs in VTM were immediately stored at −80°C until needed. Vacutainers containing clotted blood were placed on the laboratory bench for at least 1 h for further separation. Vacutainers were centrifuged at 1500 rpm for 3 mins. Sera were harvested into prelabelled 2 mL centrifuge tubes and stored at −20°C until needed.μl of each diluted sample was then pipetted into the appropriate well on the antigen-coated plate. One hundred microliters of undiluted positive and negative controls was added to their appropriate wells in duplicate. The plate was incubated for 30 mins at room temperature. Plates were then manually washed five times with deionised water and blotted dry on laboratory tissue paper after washing. Hundred microliters of conjugate was added to all wells and the plate was incubated at room temperature for 30±2 mins. Washing and blotting were repeated as described above. One hundred microliters of TMB substrate was added to all wells and incubated at room temperature for 15±1 mins. To stop the reaction, 100 μl of stop solution was added to all the wells. The optical density (OD)/absorbance value of each sample on the test plate was measured with Varioskan Lux (Thermo Scientific) at a wavelength of 650 nm.Each serum sample was singly tested for IBV antibodies using the enzyme-linked immunosorbent assay (ELISA) technique. A commercial IBV Antibody Test kit from IDEXX Laboratories Inc. (USA) which has also been used to detect IBV exposure in birds other than chickens  was used. Appropriate positive and negative controls included in the test kit were added to each plate run. The manufacturer's instructions were followed with some slight modifications. Briefly, test sera and ELISA test reagents were bought to room temperature. Prior to being assayed, a 1:500 dilution of the samples was made with manufacturer's diluent in a 2-step process. 100 2O were used as extraction positive and negative controls, respectively. Amplification of the S1 glycoprotein gene common to all IBV serotypes and the most reliable one in discriminating all IBV strains [μl thin walled PCR tube in a final reaction volume of 25 μL in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems). The reaction comprised 2X reaction buffer (12.5uL), 10 pmol IBV LC3 (0.5 uL), 10 pmol IBV LC5 (0.5 uL), RNasin Inhibitor (0.5 uL), nuclease-free H2O (5 uL), Superscript III (1 uL), and RNA extract (5 uL). Cycling conditions were reverse transcription at 50°C for 30 mins, activation step at 94°C for 2 mins, followed by 45 cycles of denaturation at 94°C for 15 sec, primer annealing at 50°C for 30 sec, extension at 68°C for 1 min, and final extension at 68°C for 5 mins. Two additional controls were added to each reaction set. These were PCR negative control (nuclease-free H2O) and PCR positive control (Ma5 vaccine).Samples from a particular source and species were processed together. RNA was extracted from all samples using the Qiagen viral RNA mini kit and following the manufacturer's instructions. Ma5 vaccine and nuclease-free H strains  was carr strains  which giAmplicons were resolved on a 1.5% agarose gel stained with ethidium bromide and visualised with a GelDoc-It®2 Imager  after 60 mins of electrophoresis in a 1% TBE buffer.OD values were transferred onto an excel worksheet. The Positive Control Means (PCX) and Negative Control Means (NCX) for each test plate were calculated . An assay was accepted to be valid when the NCX absorbance was less than or equal to 0.150 and the difference between PCX and NCX was greater than 0.075. The relative level of IBV antibody in the sample was determined by calculating the sample to positive (S/P) ratio using the following formula:S/P = Sample  (OD) − NCX  (OD)/(OD) − NCX  (OD), where S/P is sample to positive ratio, Sample (OD) is OD of test serum, NCX (OD) is mean OD of negative control, and PCX (OD) is mean OD of positive control.Serum sample with S/P ratios less than or equal to 0.20 was considered negative; S/P ratio greater than 0.20 was considered positive. Data interpretation was as provided by the manufacturer.Data was exported and analysed using SPSS 17.0.1 (SPSS Inc.). MS prevalence was calculated using the formula:Samples were collected from 29 households in the 2 communities during the 2-month period. All sampled poultry were free of respiratory symptoms at the time of sampling. The type and number of poultry kept by households differed. Three (3) types of poultry, local chickens, guinea fowls, and ducks, were often kept. Majority (59%) of households kept only local chickens while very few households (3%) kept only guinea fowls. 17% of households kept both local chickens and guinea fowls and 7% kept local chickens and duck. 14% households kept all three (3) species . The numIn all, 219 local chickens and guinea fowls were sampled during the period. The total number of samples obtained from both communities was nearly equal . HoweverNone of the 54 guinea fowl sera samples analysed tested positive for IBV antibody. Using the polymerase chain reaction method, the S1 glycoprotein gene was also not detected in any of the tracheal and cloacal swabs. The overall prevalence of IBV in guinea fowl by both serology and PCR was 0% .X2= 7.424, p value= 0.006). Of the 219 sera analysed from both poultry species 35 tested positive to IBV and 184 tested negative to IBV. The overall seroprevalence of IBV in the bird species in the two (2) communities was 16.0%.In local chickens, none of the cloacal and tracheal swabs was positive for the S1 glycoprotein gene by PCR. Serologically, however, IBV antibodies were detected in nearly a quarter (35) of the sera samples. The seroprevalence of IBV in the local chickens was determined to be 21.2% . IBV preThe local chicken and guinea fowl play an important role in the provision of cheap animal protein for many households in Africa. They are a source of lean meat and eggs and contribute to the reduction of protein malnutrition of the family , 10. A sIn our study area, we observed that local chickens and guinea fowls were kept by households in both communities but guinea fowls were more likely to be found in the Abokobi community than the Frafraha community  and the All the local chickens from Abokobi had received at least one dose of I2 vaccine against Newcastle disease virus but none of the chickens from Frafraha had been vaccinated. The vaccination follows efforts by the Ministry of Food and Agriculture's to assist households who keep village chickens in reducing losses due to Newcastle disease. The reasons for local chickens in Frafraha not being vaccinated with I2 were not pursued in this study. All the birds from both communities had not been vaccinated against IBV. Vaccination of both local and commercial chickens against IBV is not part of the scheduled vaccination of the Ghana Veterinary Services Directorate . The detThe youngest poultry sampled was more than 3 months old and therefore maternal antibodies against IBV if present would have waned before sampling. The detection of IBV antibodies in our test sera from local chickens is therefore an indication of exposure to field IBV pathogens. Our study shows that local chickens in the two communities have been exposed to IBV. This confirms reports that the virus circulates in the Ga-East district in which both communities are located . The cirIBV can be detected in the trachea during the acute stage of an infection. The virus however persists in the caecal tonsils where it is shed for several weeks in faeces after an infection . Our sampled birds showed no respiratory symptoms, thereby decreasing our chances of detecting viral RNA in the sampled trachea. Viral shedding time via faeces could have also elapsed during our sampling time because viral RNA was not detected in the cloacal swabs of all the samples including sera positive local chickens.Despite the circulation of IBV in the local chickens in the district and their intermingling with free-range birds, guinea fowls tested negative to IBV by both serology and PCR. The nondetection of IBV in guinea fowls could be attributed to a number of reasons. First of all, apart from chickens, the only other avian species with confirmed IBV infection are commercially reared pheasants , 23. HowThe IBV prevalence in local chickens reported here (21.2%) is lower compared to that reported in other West African countries by Emekpe et al.  and AdebIndigenous chickens in the Abokobi and Frafraha communities in the Ga-East district of the Greater Accra region of Ghana have been exposed to infectious bronchitis virus. Further studies are necessary to ascertain the source of infection and whether the indigenous chickens are susceptible not only to IBV, but also to infectious bronchitis disease.