Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog,  Serine proteases are a widely studied group of proteins as they play various roles in healthy and diseased tissues. Serine protease inhibitors can also modulate a series of important biological processes, such as coagulation and inflammation, making them a focus for biomedical studies ,2.Plants are remarkable sources of the serine protease inhibitor, which can be grouped into at least 10 families. The Bowman-Birk family inhibitors (BBIs) are the best studied and most widely known among them. Identified in and isolated from soybean, they were the first to often be referred to as “classical BBI”. However, subsequently multiple BBIs have been isolated from plants such as legumes and Gramineae ,4,5.Huia versabilis (HV-BBI) [Odorrana schmackeri (OSTI) [Hylarana erythraea chymotrypsin inhibitor (HECI) [Pelophyla esculentus Bowman-Birk proteinase inhibitor (PE-BBI) [1SXPPXPC, with P1 representing the inhibitory active site and X indicating that various amino acids are found in these positions. This disulfide-bridged loop is considered a trypsin inhibitory loop (TIL), which has a significant trypsin inhibitory activity [The skin of frogs is the main organ involved in their defense system, which manufactures diverse bioactive peptides that possess cytolytic pharmacological activities , and as (HV-BBI) , Hejiang(HV-BBI) , the Bowi (OSTI) , Hylaranr (HECI)  and Pelo(PE-BBI) . Generalactivity .Based on previous studies, these amphibian BBIs not only have potent protease inhibitory activities, but also exhibit an antimicrobial activity. Antimicrobial peptides (AMPs) are attractive alternatives to producing novel antibiotics. However, their susceptibility to proteases appreciably limits the potential applications of most AMPs. Therefore, a bifunctional peptide possessing both antimicrobial and protease inhibitory activities, with a low cytotoxicity, could represent an ideal template for future clinical use .Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. It was shown to possess a potent trypsin and tryptase inhibitory activity with a high specificity. Several analogues were created by rational design, and a P1 site F substituted analogue displayed a considerable and specific chymotrypsin inhibitory activity. A better antimicrobial activity was observed when P16 was replaced by Lys, and the addition of the cell-penetrating peptide Tat48–56 resulted in a peptide with a strong antifungal activity. Moreover, anti-proliferative effects on H157 and H460 were also observed.In this report, a novel peptide from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophelax plancyi fukienensis  were captured in Fuzhou City, Fujian Province, China. All frogs were adults, and skin secretion was obtained by a mild electrical stimulation on the dorsal skin surface of the frogs [Specimens of the Fukien gold-striped pond frog, he frogs . The secThe precursor encoding cDNA from the skin secretion was obtained as described previously . BrieflyThe isolation and identification of the mature peptide from the skin secretion using the RP-HPLC and LC-MS analyses were performed as outlined previously . A molec1 site, the lysine at the P1 site was replaced by a phenylalanine  which confers chymotrypsin inhibitory specificity. In the second case, the last amino acid residue, proline, was substituted with a lysine to give a positive charge and achieve a more structural similarity with the members of the Ranacyclin family, which have an antimicrobial activity . The trypsin inhibitory loop , derived from the amphibian Bowman-Birk-type protease inhibitor, is found to have a potent trypsin inhibitory activity. Thus, in the last case, a short cell-penetrating peptide Tat48-56 (RKKRRQRRR), which has been considered one of the most promising tools to improve the cellular delivery of therapeutic molecules [There are three design strategies based on the obtained parent peptide, one to alter the inhibitory specificity, one to enhance the antimicrobial activity, and one to enhance the drug delivery and cell targeting. In the first case, since synthetic work on the BBI-like peptides has focused mostly on the Polecules ,20,21, wThe novel-cloned cDNA-encoded peptide, wild-type PPF-BBI, and its analogues were synthesized by chemical synthesis using a Tribute peptide solid-phase synthesizer , as outlined previously . The synw/v) polyethylene glycol 6000 .The trypsin, chymotrypsin inhibition tests were performed as described previously . 10 µL tThe rate of hydrolysis of the substrate was monitored by measuring the rate of increase of fluorescence due to the release of 7–amino–4–methylcoumarin (AMC) at 460 nm (excitation 360 nm) in a FLUOstar OPTIMA multi-well plate reader. The inhibition curves of the trypsin/chymotrypsin inhibition assay and tryptase inhibition assay were formed as outlined before ,12.S. aureus (NCTC 10788), E. coli (NCTC 10418) and C. albicans (NCYC 1467), together with two species of resistant micro-organisms, methicillin-resistant S. aureus (MRSA) (ATCC 12493) and P. aeruginosa (ATCC 27853).The MIC and MBC of the synthetic peptides were determined as previously described , using SThe membrane permeability assay was performed as described previously . The pepThe sample peptide solutions (50 μM) were prepared in a 1 mm high precision quartz cell  with 10 mM ammonium acetate and 50% TFE in 10 mM ammonium acetate buffer respectively. CD measurements were performed at 20 °C by a JASCO J-815 CD spectrometer  across the wavelength range of 190–250 nm. The scanning speed was 100 nm/min, the bandwidth was one nm, and the data pitch was 0.5 nm. The CD spectra were further analysed using the online software, BeStSel , and the® HTB-177™), H838 (ATCC® CRL-5844™), and H23 (ATCC® CRL-5800™), as well as other cancer cell lines: HT-29 (ATCC® HTB-38™), PC-3 (ATCC® CRL-1435™), U251MG (ECACC-09063001), and the normal human dermal microvascular endothelium cell line HMEC-1 (ATCC® CRL-3243™). The anti-metabolite 5-fluorouracil (5-FU) was utilized as the positive control group.The MTT assay was carried out as described in a previous study , using aThe haemolytic activity of each peptide was measured by incubating a range of final peptide concentrations from 512 to 1 µM in a two-fold dilution in a 2% suspension of horse erythrocytes, as described in a previous study .The data of all the bioactive assays were statistical analyzed using Prism 6 . The data points represent the average of three independent experiments with the error bars representing the standard error of the mean (SEM).Pelophlax plancyi fukienesis was analysed by LC-MS. The retention time of the fragmentation indicated in From a skin-derived cDNA library, a cDNA encoding a biosynthetic precursor of PPF-BBI was consistently and repeatedly cloned . The cru1 site, F8-PPF-BBI, was aimed to produce a chymotrypsin inhibitory peptide. The substitution of a lysine at the position 16 of the native peptide (K16-PPF-BBI) enhanced the net positive charge, which might improve the ability to interact with the cell membrane. The Tat sequence was added at the N-terminus of the typical 11-mer trypsin inhibitory loop structure to increase the membrane penetration effect. All the analogues were chemically synthesized, purified by RP-HPLC and analysed by MALDI-TOF.Three analogues were designed based on the natural peptide PPF-BBI . Briefly8-PPF-BBI and K16-PPF-BBI revealed a broad negative band with the minimum around 200 nm, typical of an unfolded peptide in equilibrium with a β-sheet structure [8-PPF-BBI and K16-PPF-BBI displayed the same red shift trend of the negative minimum. Since the spectra displayed a broad minimum spanning the region 200–210 nm and did not show positive bands above 210 nm, this suggests that the conformation of peptides is likely to consist of a mixture of secondary structures of β-sheet structure and random coil, which is consistent with previous studies [PPF-BBI and the analogue peptides were successfully synthesized, impurities were removed by HPLC, and their identity was confirmed by MALDI-TOF. The secondary structures of all peptides were determined by circular dichroism spectroscopy . PPF-BBItructure , except  studies ,24,25,2616-PPF-BBI exhibited a potent trypsin inhibitory activity. However, K16-PPF-BBI had a more potent inhibitory effect against tryptase than the parent peptide did. In addition, F8-PPF-BBI only displayed a strong chymotrypsin inhibitory activity and lost the trypsin inhibitory activity. Interestingly, the trypsin inhibitory loop (TIL) and Tat-loop kept the trypsin inhibition activity but did not exhibit a tryptase inhibitory activity  to generate the Morrison plots. All of the progress curves and corresponding Morrison plots in the presence of each peptide of different concentrations are shown in activity .16-PPF-BBI displayed a mild activity against the tested microorganisms, although K16-PPF-BBI exhibited a better bioactivity than the parent peptide against S. aureus and C. albicans. However, although its activity against the other microorganisms was similar to the parental peptide, Tat-loop showed a much more potent activity against C. albicans. The component parts of Tat-loop (separate Tat peptide and the TIL) were also tested, but had little activity on their own. To sum up, Tat-loop showed a potent activity against C. albicans, as well as exhibiting slightly more activity against MRSA and P. aeruginosa. K16-PPF-BBI exhibited the best activity against S. aureus and was slightly better than PPF-BBI against all of the others except E. coli.The antimicrobial activity of PPF and its analogues was tested against a representative set of microorganisms . Both PPC. albicans was due to it impacting its membrane permeability. However, Tat-loop did not cause any membrane permeabilization, even at high concentrations  and other cancer cell lines , and were also tested on a human normal cell (HMEC-1) a. Among Pelophylax plancyi fukienensis. In addition, we also examine the bioactivity of three rationally designed analogues of PPF-BBI, F9-PPF-BBI, K16-PPF-BBI and Tat-loop.In this study, we report the identification and bioactivity evaluations of PPF-BBI, a novel Bowman-Birk type protease inhibitor from the skin secretion of the Fukien gold-striped pond frog, 1 position residue can fit into the S1 pocket of protease. Based on previous reports, Lys as the P1 position is optimal for trypsin inhibition, and Phe is optimal for chymotrypsin [1 position, with Phe leading to the elimination of the trypsin inhibition and giving rise to a chymotrypsin inhibitory activity. In the tryptase activity assay, PPF-BBI displayed a mild potency toward tryptase with a Ki value of 30.52 µM. K16-PPF-BBI shows a three-fold better inhibition with a Ki value of 9.67 µM, but intriguingly, Tat-loop and TIL lost their inhibitory activity against tryptase, even though they retained their trypsin inhibitory activity. Indeed, this contradicts a previous study [16-PPF-BBI showed here, the substitution of Lys at the C-terminus improved the tryptase inhibitory activity, indicating that the C-terminal extensive residue might contribute to the binding between the BBI peptide and tryptase. Therefore, the lack of such an extension of TIL and Tat-loop could eliminate the affinity to the reactive pocket of tryptase, so that both cannot produce any inhibitory activity.Like other BBI-type peptides, PPF-BBI has potent protease inhibitory activities. The specificity of inhibition is determined by whether the Potrypsin ,27. Simius study , which dus study . As the 16-PPF-BBI shared a high sequence similarity with Ranacyclin members, and they were also found to have moderate effects on the tested microorganisms, as was expected. However, K16-PPF-BBI was shown to have a better effect, which is possibly because one more lysine increases its positive charges, and it is easier to get it close to the negative groups of the cell membrane.The mode of action of Ranacyclins is different from most known positively charged antimicrobial peptides. They bind and insert into both zwitterionic and negatively charged membranes, and they presumably form transmembrane pores without bacteria wall damage. Indeed, it has been reported that Ranacyclins E and T have a great potential as antimicrobials . PPF-BBITat, a cationic-rich cell-penetrating peptide derived from the HIV protein, has been used to conjugate with other compounds to enhance the cell penetrating activity. In the previous study, the design of Tat-fusion biopeptides demonstrated a remarkable improvement on their biological activities ,21. In tC. albicans with MIC of 4 µM) of Tat-loop. However, this does not appear to be due to its impact upon the membrane, as treatment with this peptide does not induce changes in membrane permeability even at high concentrations . Using the principle of plant protease inhibitors, it would be pertinent to evaluate the antifungal activity of Tat-loop, possibly because it might interfere with the trypsin-mediated activation of the chitin synthase zymogen and further affect the process of cell wall development [Interestingly, this is reflected in the significant increase of potency against the tested strains (especially elopment .9-HECI, exhibited great anti-proliferative potency against H157, PC-3 and MCF-7. Also, PE-BBI was reported to have an inhibition effect on several colon cancer cell lines [In the report by Zhang et al. , a Bowmall lines . In our 9-PPF-BBI and K16-PPF-BBI initiated a slight conversion from a random coil into a β-structure in the TFE buffer due to assisting the folding of the secondary structure [In addition, PPF-BBI, Ftructure . Howevertructure . We assutructure ,25,34.C. albicans, which also results from its inhibition of trypsin, which might have potential towards fungal diseases. However, the mechanism by which Tat-loop impacts upon C. albicans is unclear, and further research is required to determine how this peptide exerts its impact upon this fungus.In summary, PPF-BBI is a naturally occurring peptide with a remarkable trypsin and tryptase inhibitory activity, as well as a moderate antimicrobial activity. This provided the basis for the rational design of further multifunctional protease inhibitors. Moreover, this is the first report investigating the addition of a cell-penetrating peptide to an amphibian skin-derived protease inhibitor. Tat-loop has high potency against


Ulteriorly, the inhibition type and kinetics of CAG towards UGT1A8 and UGT2B7 were evaluated depending on the initial screening results. Data fitting using Dixon and Lineweaver–Burk plots demonstrated that CAG competitively inhibited UGT1A8 and noncompetitively inhibited UGT2B7. From the second plot drawn with the slopes from the Lineweaver–Burk plot versus the concentrations of CAG, the inhibition constant (Ki) was calculated to be 0.034 μM and 20.98 μM for the inhibition of UGT1A8 and UGT2B7, respectively. Based on the [I]/Ki standard , it was successfully predicted here that an in vivo herb–drug interaction between AST/CAG and drugs mainly undergoing UGT1A8- or UGT2B7-catalyzed metabolism might occur when the plasma concentration of CAG is above 0.034 μM and 20.98 μM, respectively.As one of the main active ingredients from Radix Astragali (RA), orally dosed astragaloside IV (AST) is easily transformed to sapogenin-cycloastragenol (CAG) by deglycosylation in the gastrointestinal tract. Because the potential adverse effects of AST and CAG remain unclear, the present study in this article was carried out to investigate the inhibition effects of AST and CAG on UDP-glucuronosyltransferases (UGTs) to explore potential clinical toxicity. An in vitro UGTs incubation mixture was employed to study the inhibition of AST and CAG towards UGT isoforms. Concentrations of 100 μM for each compound were used to initially screen the inhibitory efficiency. Deglycosylation of AST to CAG could strongly increase the inhibitory effects towards almost all of the tested UGT isoforms, with an IC Radix Astragali (RA), originating from the dried root of Astragalus membranceus (Fisch.) Bge. and A. membranceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao  represents the mean steady-state Cmax value following the administration of the highest proposed clinical dose /cal dose . When CAAlthough speculation on the clinical relevance of in vitro observations of inhibition of glucuronidation are relatively common, appropriate mechanistic studies attempting to link in vitro inhibition with influence on AUC in humans are rare. In fact, high risk occurs only when the AUCi/AUC ratio is above 5; however, observed AUCi/AUC ratios for glucuronidated drugs co-administered with UGT inhibitors are typically less than 2, thus indicating a low possibility of inhibitory drug-drug interactions caused by UGTs . In view50 of CAG towards UGT1A8 and UGT2B7 was calculated to be 0.84 μM and 11.28 μM, respectively. The inhibition constants (Ki) were calculated to be 0.034 μM and 20.98 μM for UGT1A8 and UGT2B7, respectively. The experimental data will be of considerable referential importance when RA or AST/CAG is co-administered with drugs mainly metabolized by UGT1A8 or UGT2B7 for consequent excretion.In conclusion, the present study investigated the inhibitory potential of AST and CAG towards 11 UGT isoforms, to predict the possible herb-drug interactions between RA/AST and co-administrated drugs. The ICd-glucuronide (4-MUG), 7-hydroxycoumarin, Tris-HCl, and uridine-50-diphosphoglucuronicacid (UDPGA)  were purchased from Sigma-Aldrich . Recombinant human UGT supersomes  expressed in baculovirus-infected insect cells were obtained from BD Gentest Corp. . All other reagents were of high-performance liquid chromatography (HPLC) grade.AST and CAG were purchased from Tianjin Yousi Pharma Ltd. , with the purity both above 98%. 4-Methylumbelliferone (4-MU), 4-MU-b-2, 50 mM Tris-HCl buffer (pH = 7.4), and various concentrations of 4-MU. 4-MU was used as a non-selective substrate of UGTs. There was a 5 min pre-incubation step at 37 °C before the reaction was initiated by the addition of UDPGA. 4-MU and inhibitors were previously dissolved in DMSO, and the total concentration of DMSO was 1% (v/v). The reactions were continued at 37 °C for 120 min for UGT1A1, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17, 75 min for UGT1A3, and 30 min for UGT1A6, UGT1A7, UGT1A8, and UGT1A9, respectively. Reactions were terminated by the addition of 100 μL of acetonitrile with 7-hydroxycoumarin (100 μM) as the internal standard. The incubation mixtures were then centrifuged at 12,000× g for 10 min. The in vitro incubation experiment was carried out according to previously literature . In brie® BEH C18  at a flow rate of 0.2 mL/min and an UV detector at 316 nm. The mobile phase consisted of ultrapure water containing 0.5% formic acid (v/v) (A) and acetonitrile (B). The following gradient condition was used: 0–3.5 min, 10%–65% B; 3.5–4.0 min, 65% B; 4.0–9.0 min, 10% B. The retention times for 4MUG, 7-hydroxycoumarin  and 4MU were 2.6, 3.2, and 3.5 min, respectively.An amount of 2 μL of supernatant was injected into the UPLC system for analysis. A Waters ACQUITY UPLC System equipped with an UV detector was used to analyze the samples, and the separation of all compounds was carried out using an ACQUITY UPLC2 over 99% (1/r2). We force the calibration equation through zero because we assume no HPLC response when no metabolite exists. The fitting equation was y = 0.0282x. The detection and quantification limits were obtained via signal-to-noise ratios of 3:1 and 10:1, respectively. The LOD and LOQ were 0.015 μM and 0.03 μM, respectively. The accuracy and precision of the back-calculated values for each concentration were less than 5%. All experiments were separately performed in duplicate three times. The determination of IC50 was calculated using Probit analysis in SPSS11.5 . The Student’s t-test was adopted at a significance level of p < 0.05 to determine statistically significant differences among experimental groups.To calculate the standard curve, 0.1–100 μM 4-MUG was used by drawing the peak area ratio of 4-MUG/internal standard towards the concentration range of 4-MUG, with a linear correlation coefficient r50) values were determined using various concentrations of CAG . Inhibition constant (Ki) were determined utilizing various concentrations of 4-MU  in the absence (control) and presence of different concentrations of CAG . Dixon and Lineweaver–Burk plots were employed to determine the inhibition type, and the second plot of the slopes from the Lineweaver–Burk plot versus the compound concentrations was utilized to calculate the Ki value.The inhibition type and Kinetics were determined for the inhibition of CAG towards UGT1A8 and UGT2B7. The half inhibition concentration (ICIn vitro–in vivo extrapolation (IVIVE) was performed using the following equation:in vivo is the in vivo exposure concentration of CAG, and the Ki value is the in vitro inhibition constant.The terms are defined as follows: AUCi/AUC is the predicted ratio of the in vivo exposure of the xenobiotics or endogenous substances with or without the co-exposure of CAG. [I]


Dalbergia parviflora with vast structural diversity were screened for inhibitory activity against mushroom and murine tyrosinases using l-DOPA as the substrate. Among the flavonoids tested, only four—khrinone (5), cajanin (9), (3RS)-3′-hydroxy-8-methoxy vestitol (21), and -3,8-dihydroxy-9-methoxy pterocarpan (27)—reacted with mushroom tyrosinase, with IC50 values of 54.0, 67.9, 67.8, and 16.7 μM, respectively, and only compound 27 showed inhibitory activity against murine tyrosinase. With cell-based assays, only compounds 9 and 27 effectively inhibited melanogenesis in B16-F10 melanoma cells , at a concentration of 15 μM, without being significantly toxic to the cells. However, the crude extract of D. parviflora and some of the flavonoid constituents appeared to increase melanin production in B16-F10 cells, suggesting that there are flavonoids with both inhibitory and stimulatory melanogenesis in the crude extract. Studies on the correlation between the enzyme-based and cell-based assays showed that only the flavonoids with IC50 values below 50 μM against mushroom tyrosinase could inhibit the mammalian tyrosinase, and thus, reduce melanogenesis in B16-F10. Flavonoids with the IC50 values greater than 50 μM, on the other hand, could not inhibit the mammalian tyrosinase, and had either no effect or enhancement of melanogenesis. In conclusion, the tyrosinase enzyme from mushroom is not as selective as the one from mammalian source for the enzyme-based melanogenesis inhibitory screening, and the mammalian cell-based assay appears to be a more reliable model for screening than the enzyme-based one.Twenty-seven flavonoids isolated from  In plant-derived foods, tyrosinase catalyzes the oxidation of phenolic compounds to their corresponding quinones, and is responsible for the enzymatic browning of fruits and vegetables [Tyrosinase EC 1.14.18.1) is the key enzyme involved in melanin biosynthesis in living organisms. In animal cells, tyrosinase oxidizes .14.18.1 getables ,3,4,5.Dalbergia parviflora, a Thai folk medicine traditionally used as a blood tonic and menstruation normalizer. More than 60 flavonoids were isolated and identified, including 22 isoflavones, 12 isoflavanones, 10 isoflavans, six flavonones, four flavanonols, and six pterocarpans [D. parviflora has also been reported [Mushroom tyrosinase has been widely used as a substitute for mammalian tyrosinase to screen for tyrosinase inhibitors as it is inexpensive and commercially available in a purified form . Severalocarpans . The strD. parviflora were tested for their ability to inhibit mushroom and murine tyrosinases using l-DOPA as the substrate. The inhibition of mushroom tyrosinase was then compared to the inhibition of crude murine tyrosinase extracted from B16-F10 melanoma cells. Finally, the effects of these compounds on the overall melanin production in B16-F10 melanoma cells were studied to observe the correlation between the results of the enzyme-based and cell-based assays.In this study, crude extract and 27 flavonoids prepared and isolated from the heartwood of D. parviflora, grouped into isoflavones, flavanones, isoflavanones, isoflavans, and pterocarpan, are shown in o-diphenolase inhibitory activity of mushroom tyrosinase using l-DOPA as the substrate. A total of 11 active inhibitors from the preliminary screening were then determined for their 50% of inhibition (IC50) values, and the results are summarized in The structures of the 27 flavonoids isolated from 1–9), only two compounds with a hydroxyl group at the C-2′ position of ring B—khrinone B (5) and cajanin (9)—exhibited o-diphenolase inhibitory potential, with IC50 values of 54.0 ± 6.0 and 67.9 ± 6.2 μM, respectively. The other five isoflavones—namely formononetin (1), calycosin (2), biochanin A (3) 3′-O-methylorobol (6), and khrinone C (7)—did not show inhibitory activity on mushroom tyrosinase. In contrast to a previous report, calycosin has been shown to be a monophenolase inhibitor of mushroom tyrosinase with an IC50 value of 38.4 μM. In addition, calycosin reduced melanin biosynthesis in murine melan-a melanocytes [R,S)-3′-hydroxy-8-methyoxy vestitol (21), an isoflavan with hydroxyl groups at the R2′and R3′ positions of ring B, showed inhibition with an IC50 value of 67.8 ± 5.8 μM. Pterocarpan (27) was the best mushroom tyrosinase inhibitor among the 27 flavonoids tested, with an IC50 value equal to kojic acid . Moreover, the positive control, oxyresveratol, exhibited the strongest inhibitory effect on mushroom tyrosinase with an IC50 of 0.19 ± 0.06 μM. The crude extract of D. parviflora exhibited tyrosinase inhibitory activity with an IC50 of 2.6 ± 0.4 μg/mL.From the nine isoflavones -8-demethylduartin (23) contained 7,8-dihydroxy groups. However, the inhibitory activities of khrinone B (5) and cajanin (9) suggested that not only the 6,7- or 7,8-dihydroxy groups of ring A, but also the hydroxyl group at the C2′ position of ring B of isoflavone or isoflavanone skeletons, might play important roles in the expression of o-diphenolase inhibitory activity. The inhibitory activities of (3RS)-3′-hydroxy-8-methoxy vestitol (21) and -3,8-dihydroxy-9-methoxy pterocarpan (27) supported the importance of both the hydroxyl group at the C3′ position on ring B of isoflavans and pterocarpan for o-diphenolase inhibitory activity.In the series of flavonoids in this study, only (3S)-8-demethylduartin (23), which could be a potent tyrosinase inhibitor. However, after pre-incubation of (3S)-8-demethylduartin (23) with the enzyme in the absence of the substrate (l-DOPA), an orange metabolite with an optical density at 475 nm was observed. This observation suggests that (3S)-8-demethylduartin (23) can act as a chromogenic substrate analog for mushroom tyrosinase. The other flavonoids that were found to be chromogenic substrate analogs include genistein (4), tectorigenin (8), and (2S)-naringenin (25). These results are comparable to a previous report in which resveratrol, a phenylpropanoid, was oxidized by mushroom tyrosinase and converted to a newly oxidized compound which was observed at 475 nm and became a tyrosinase inhibitor [l-tyrosine or l-DOPA, generating false inhibition or activation effects [In this study, the 7,8-dihydroxy groups are only present in -3,8-dihydroxy-9-methoxy pterocarpan (27) showed a weak inhibitory effect on the o-diphenolase activity of murine tyrosinase with an inhibition of 29.2 ± 2.9% (50 = 39.9 μM) and 99.4 ± 0.9% (IC50 = 0.88 ± 0.16 μM), respectively.The inhibition of murine tyrosinase was studied using the flavonoids at a concentration of 200 μM. The results revealed that among the 27 flavonoids, only 2 ± 2.9% . The cru(Agaricus bisporus) tyrosinase. Although these results may highlight that mushroom tyrosinase can be used for anti-tyrosinase activity screening, it does not appear to be sufficiently accurate to measure the potential of the active substance as a whitening agent. A more appropriate method would be to use the mammalian tyrosinase inhibition assay and melanogenesis inhibition results from the cell-based assay. However, mushroom tyrosinase is still a suitable screening tool to measure the anti-browning activity of plant-derived foods in the food industry.Notably, the flavonoids exhibited stronger effects on mushroom tyrosinase than murine tyrosinase activity. In fact, almost all studies conducted on tyrosinase inhibition have used the commercially available champignon mushroom Murine B16-F10 melanoma cells were used as a model to examine the effects of the 27 flavonoids on cell viability and melanogenesis . The mel13, 15, and 26 exhibited cytotoxicity against melanoma cells at a concentration of 15 μM with the cell viability decreasing to approximately 70–80% of the control. Compounds 5, 14, and 21 showed toxicity at a concentration of 30 μM with a cell viability of 60%. The highest cytotoxicity was found in compound 27, with a cell viability of 14% at 30 μM, but no toxicity was exhibited at 15 μM. Compounds 8, 16, 19, and 22 increased the quantity of melanoma cells with a cell viability of 120–140% compared with the control at a concentration of 15 μM. The inhibitory effect of melanogenesis was found in compounds 9 and 27. Although compound 27 exhibited 60% inhibition of melanogenesis at 15 μM without affecting the cell viability, it showed cytotoxicity at 30 μM (9 significantly decreased the cellular melanin content without affecting the cells at both concentrations.Compounds at 30 μM . Only co22), which exhibited a three-fold increase in melanin content with respect to the control  and (3S)-8-demethylduartin (23) revealed that the 7,8-dihydroxy structure in the isoflavans was associated with melanogenesis inhibition. The R8-OMe substitution in duartin (22) had a strong effect on melanogenesis stimulation. The comparison of chemical structures between (3RS)-3′-hydroxy-8-methoxy vestitol (21) and duartin (22) also revealed that the 2′,3′-dihydroxy structure was associated with an inhibition of mushroom tyrosinase. Duartin (22), associated with 2′-methoxy structure, exhibited the strongest melanogenesis stimulation and loss of mushroom tyrosinase inhibitory activity.A previous report indicated that quercetin and naringenin stimulate melanogenesis in cultured murine B16-F10 melanoma cells. Kubo et al. suggested that the improvement of melanin production by quercetin may be due, in part, to melanocytotoxicity , whereasA. bisporus has been reported to be a soluble cytosolic enzyme containing two different subunits, referred to as heavy (H) and light (L), with molecular weights of 43 and 13.4 kDa, respectively. In aqueous solution, the predominant form of the mushroom tyrosinase is the quaternary L2H2 structure with a molecular weight of 120 kDa, whereas the active form appears to be L2H with an apparent molecular weight of 69 kDa [50 lower than 50 μM. In other words, most flavonoids are likely to stimulate melanogenesis despite inhibiting mushroom tyrosinase activity.All tyrosinases have a binuclear type three copper center within their active site in common. However, no common tyrosinase protein structure has been found to occur across all species ,27,28,29f 69 kDa . In contf 69 kDa . A studyl-phenylalanine (l-DOPA), kojic acid, oxyresveratrol, and mushroom tyrosinase were purchased from Sigma-Aldrich . The 27 flavonoids from D. parviflora were obtained from Kaoru Umehara [3,4-Dihydroxy- Umehara ,11.l-DOPA, and tyrosinase enzyme solutions were dissolved in 50% (v/v) dimethyl sulfoxide (DMSO) and 20 mM phosphate buffer solution (PBS) of pH 6.8, respectively. The o-diphenolase activity of mushroom tyrosinase was performed in 96-well plates using a modified method of that performed by Likhitwitayawuid and Sritularak [l-DOPA was added, and the mixture was incubated for 20 min at 25 °C. During the reaction, l-DOPA was converted to dopachrome, which resulted in a change in color from colorless to orange. This change was measured through absorbance at 475 nm. Instead of the sample solution, 50% DMSO was used as the control. The reaction mixture without enzyme served as a blank. The inhibitory activity of the sample was expressed as the concentration that inhibits 50% of enzyme activity (IC50).The test samples, itularak . The reaMus musculus (mouse) melanoma cell line, B16-F10 , was cultured in complete Dulbecco′s Modified Eagle′s Medium  containing 10% fetal bovine serum, 25 mM glucose, 4 mM l-glutamine, 1 mM sodium pyruvate, 10 μg/mL penicillin, and 10 μg/mL streptomycin, at 37 °C, with 5% carbon dioxide (CO2) in a humidified atmosphere.g for 15 min at 4 °C to remove the cell debris. The supernatant was dialyzed against 20 mM sodium phosphate buffer (pH 6.8) to remove the Triton X-100. The dialyzed solution was used as a source of crude murine tyrosinase enzyme. The solution contained 1 mg/mL protein, which was determined using a protein assay kit, based on the Bradford method . The inhibitory activity was determined as described above for mushroom tyrosinase, but the incubation time was optimized at 2 h and 37 °C.B16-F10 melanoma cells were grown to 100% confluence on 10 cm dishes. The trypsinized cells were washed with cold phosphate buffer  and then disrupted in 1 mL of the buffer containing 1% Triton X-100. The cells were lysed by vortex and incubated at 4 °C for 1 h to solubilize the tyrosinase enzyme. The solution was centrifuged at 13,000× Cell viability and melanin content assay were performed in quadruplicate in at least three independent experiments. Cell viability was taken as the reducing power of the living cells that reduce resazurin-based solution  to resorufin, which results in a bright red color and is highly fluorescent. Briefly, B16-F10 cells were cultured in a 96-well plate at a concentration of 10,000 cells/0.1 mL/well. After incubating for 24 h, equal volumes of fresh medium containing various concentrations of the test samples were added to the cells and were incubated for 72 h. Cells were then washed with phosphate buffer, and then 0.1 mL of PrestoBlue™ solution in RMPI 1640 serum free media (1:10) was added to each well. After an hour, the fluorescence was quantified using spectrofluorophotometry .g for 5 min. Finally, the supernatant was transferred to a 96-well plate and the relative melanin content was detected at 405 nm using a micro plate reader. Kojic acid and oxyresveratrol were used as positive controls.The total melanin content, produced both extracellularly and intracellularly, was determined as follows: B16-F10 cells were seeded onto a 24-well plate at a concentration of 50,000 cells/0.5 mL/well and were incubated for 24 h, followed by treatment with either the test compounds or the control (0.5% DMSO), and then incubated for 72 h. For extraction, the total produced melanin and 0.5 mL of 2 N NaOH containing 20% DMSO were added directly to the cell culture medium, mixed by pipetting and then incubated at room temperature for 1 h. The lysed cell extract was centrifuged at 13,000× Dalbergia parviflora used in this test, -3,8-dihydroxy-9-methoxy pterocarpan (27) and cajanin (9) showed clear inhibitory activities on both methods. However, some flavonoids, particularly duartin (22), showed induction of cellular melanin synthesis without significant effect on the enzyme activity. The mechanism involved in the observed action is still not clear.In conclusion, the results from the current study suggest that the enzyme-based assay using murine tyrosinase from B16-F10 melanoma cells is more appropriate than the mushroom tyrosinase for screening of natural compounds with potential whitening effects. The B16-F10 cells are also good for a cell-based assay for evaluating the direct effect of test compounds on melanin synthesis. Among all flavonoids from


Lactobacillus bulgaricus LB6 were optimized to increase the angiotensin converting enzyme  inhibitory activity by Box–Behnken design of response surface methodology. Incubation temperature, whey powder, and calcium lactate had significant effects on ACE inhibition rate and viable counts of LB6 during incubation. The results showed that optimal conditions of fermentation were found to be 37.05 °C, 0.8% (w/w) whey powder and 0.50% (w/w) calcium lactate. ACE inhibition rate increased significantly from 71.04 ± 0.37% to 83.31 ± 0.45% and the viable counts of Lactobacillus bulgaricus LB6 reached to 8.03 × 107 cfu·mL−1 under the optimal conditions, which approached the predicted values 83.25% and 8.04 × 107 cfu·mL−1. The optimal fermentation conditions can be a good reference for preparing ACE inhibitory peptides from goat milk.In the present study, the incubation conditions of goat milk fermented by  Hypertension is a major risk factor for myocardial infarction, heart failure, stroke, aneurysm, arteriiosclerosis, renal disease, and peripheral arterial disease and it may lead to chronic kidney disease as well ,2,3. SixACE, also known as Kinase II, is a metalloproteinase containing two zinc-catalytically active sites , which cACE inhibitors can inhibit ACE activity. Since the side effects such as dry-cough, pruritic rash, and taste disorders occurred when using synthetic ACE inhibitors ,12, studLactobacillus casei NK9 and Lactobacillus fermentum LF. Sixteen peptides in fermented milk beverage made from caprine milk were identified by Quiro’s et al. .RSM (Response surface methodology) was carried out to determine the optimum concentrations of the three significant factors for fermented goat milk, incubation temperature (A), whey powder (B), and calcium lactate (C). The factors and levels are given in 1 and R2 are corresponding expected value of ACE inhibition rate and viable counts of LB6. A, B and C are the coded values of the independent variables, incubation temperature, whey powder, and calcium lactate, respectively.The data were analyzed to get quadratic regression model by using Design Export based on the data from the Box–Behnken experiment. A multiple regression equation correlating the response function with the independent variables could be obtained as:Analysis of variance (ANOVA) is used to determine whether the model is valid according to the significance. ANOVA for response surface of ACE inhibition rate and viable counts of LB6 were shown in p = 0.0007 < 0.001) and the lack of fit was insignificant (p = 0.3878 > 0.05). It revealed the effectiveness of regression analysis, which suggested that the regression model could be used to fit the effect of three factors on ACE inhibition rate. The difference between adjusted R-squared and predicted R-squared was 0.16, within the value of 0.2, which implied that the model was highly significant. Furthermore, the value of determination coefficient (R2 = 98.24%) was calculated. It suggested that a 98.24% response to the ACE inhibition rate was caused by changing in the concentration of A, B, and C and their interactions.As shown in p-value was less than 0.05, which was significant in p = 0.0939 > 0.05) was insignificant. Therefore, the model equation was corroborated to be a suitable model to describe the value of the viable counts for LB6. The value of determination coefficient, which was 98.27%, indicated that only 1.73% of the variability in the viable counts of LB6 could not be explained by the predicted equation of model. AB and BC showed weak mutual interactions on the effect of viable counts for LB6, while the interaction of AC was significant (p = 0.0488 < 0.05).The ANOVA showed the model for viable counts of LB6 with a pAB = 0.5161 > 0.05, pAC = 0.2995 > 0.05, pBC = 0.6435 > 0.05). Therefore, the effect of one factor on ACE inhibition rate was dependent on the level of another one. The quadratic main effects of incubation temperature, whey powder, and calcium lactate were significant , which suggested that there was not a simple linear correlation between the variables and ACE inhibition rate.Two-dimensional contours revealed that ACE inhibition rate changed with the changes of temperature, whey powder, and calcium lactate and their corresponding three-dimensional response surface were generated to determine the interaction of the three variables on the corresponding variables better . As showL. bulgaricus LB6 8.03 × 107 cfu·mL−1 were obtained at 37.05 °C, 0.8% whey powder, and 0.5% calcium lactate as predicted, respectively. The design of verification experiment was dependent on the optimization results . The results demonstrated that the ACE inhibition rate and viable counts of LB6 were 83.31 ± 0.45% (n = 3) and 8.03 × 107 cfu·mL−1 (n = 3) under the optimum conditions. There was no significant difference from the predicted value, indicating suitability of the model. The control had values of ACE inhibition rate as 71.04 ± 0.37% and viable counts as 4.53 × 107 cfu·mL−1 at 37 °C without addition of whey powder and calcium lactate. The ACE inhibition rate was improved by 12.27% using RSM optimization compared to the control. The viable counts of LB6 was increased significantly from 4.53 × 107 cfu·mL−1 to 8.03 × 107 cfu·mL−1.The maximum responses values of ACE inhibition rate 83.25% and the viable counts of It had been reported that great quantities of optimum methods were used to release more ACE inhibitory peptides and the approaches to enhance ACE inhibitory activity in fermented products have recently received considerable attention. B vitamins were added to the soybean milk fermentation medium and the ACE inhibitory activity had significant improvements compared to control. The addition of B vitamins enhanced protein hydrolysis degree, which resulted in more bioactive peptides being released . PrebiotL. bulgaricus and the activity of proteolytic enzyme. L. bulgaricus LB6 exhibited extremely poor growth with a weak metabolic activity when the temperature was low, causing low values of ACE inhibition rate and viable counts of LB6. With the increase of temperature, L. bulgaricus grew quickly and the enzyme activity increased gradually. Therefore, the activity of LB6 and the ACE inhibition were enhanced. The activity of proteolytic enzymes was destroyed with temperature elevation, causing the gradual decrease of ACE inhibition rate.Temperature was testified to be a significant factor to fermented milk, which could owe to the influence on the growth of Lactobacillus casei with increasing concentrations of whey powder [Whey powder has been used to improve production of ACE inhibitory peptides in a suitable concentration. The viable counts of LB6 and ACE inhibition rate in fermented goat milk increased first and then decreased as the concentration of whey powder increasing. However, the ACE inhibition rate gradually lessened in the cow milk fermented by y powder , which mL. casei and Bifidobacterium bifidum were studied by Gonzalez et al. [Low concentrations promote ACE inhibitory activity, while high concentration suppression of ACE inhibitory activity is also applicable to calcium lactate. It was reported that calcium dihydrogen phosphate was added when preparing rapeseed ACE inhibitory peptides by liquid-state fermentation conditions and the ACE inhibition rate was greatly improved . The effz et al. . It was L. bulgaricus fermentation.The ACE inhibition rate and viable counts of LB6 can be significantly increased by optimizing incubation conditions of fermented goat milk using RSM. The ACE inhibition rate improved from 71.04 ± 0.37% to 83.31 ± 0.45% in present study, which provides a reference for improving the production of ACE inhibitory peptides by Kluyveromyces marxianus, was investigated by Li et al. [v/v) inoculum level and rotation speed of 189 rpm. ACE inhibition rate reached 81.23 ± 2.69% under optimal conditions. It is lower than the result of this study, which may be due to that Lactobacillus proteolytic enzyme is superior to yeast. Pan et al. [Lactobacillus helveticus LB10 as starter culture to ferment milk under optimal conditions, which were 4% (v/w) inoculum, initial pH 7.5 of medium at 39.0 °C. Strains with high ACE inhibitory activity were screened by Chen et al. [L. helveticus was lower than that of L. bulgaricus under the same fermentation conditions, which was consistent with the results of this paper.The effect of fermentation conditions on the ACE inhibitory activity in milk fermented by yeast, i et al.  using RSn et al.  found thL. bulgaricus LB6 was provided by the college of Food and Biological Engineering, Shaanxi University of Science & Technology . The starter cultures were stored at −20 °C in freeze-dried powder. L. bulgaricus LB6 was activated successively three times in rehydrated Man Rogosa Sharpe broth  with 5% inoculum at 37 °C for 24 h prior use as starter culture to ferment goat milk.L. bulgaricus LB6 was inoculated into reconstituted skim goat milk pasteurized with 5% inoculum and fermented at 37 °C for 12 h.Starter culture containing g for 15 min to obtain the corresponding whey fractions. The supernatants collected were filtered through a Xinhua filter and used to determine their ACE inhibitory activity. ACE inhibitory activity was measured by a spectrophotometric assay according to the method of Cushman and Cheung (1971) with modifications. Added 80 μL of each sample to 200 μL sodium borate buffer  containing NaCl (0.30 mol·L−1) and hippuryl-histidyl-leucine (HHL) (5 mmol·L−1). ACE  was added and incubate the reaction mixture at 37 °C for 30 min. 250 μL 1 mol·L−1 HCl was added to terminate the reaction. Hippuric acid formed was extracted by 1.7 mL ethyl acetate and evaporated at 120 °C for 30 min. The sample was dissolved into 2 mL deionized water after cooling at room temperature. The absorbance at 228 nm was measured in triplicate using UV-vis spectrophotometer .The whey fraction from the fermented goat milk was used to measure the ACE inhibitory activity. Aliquots of the fermented goat milk were collected, vigorously stirred, and centrifuged at 5000× 1 is absorbance without the whey fraction, X2 is absorbance without ACE, and X3 is the absorbance in the presence of both ACE and the whey fraction.ACE inhibition rate were determined by the following equation:w/v, NaCl) containing 0.1 g·L−1 peptone  and spread on MRS agar plates. The dilution in triplicate was incubated for 48 h at 37 °C. Results were expressed as colony forming units per milliliter (cfu·mL−1) of fermented milk by manual counting.Fermented goat milk was diluted serially by saline water  was used for the analysis of the experimental data obtained and quadratic regression equation was established to analyze the response surface contour and surface plots.L. bulgaricus LB6 were optimized by RSM to increase the production of ACE inhibitory peptides. The results showed that the maximum ACE inhibition rate was 83.31 ± 0.45% in the 0.8% (w/w) whey powder and 0.50% (w/w) calcium lactate at 37.05 °C. The viable counts of L. bulgaricus LB6 was reaching to 8.03 × 107 cfu·mL−1 under the optimal conditions. Both ACE inhibition rate and viable counts were close to the predicted values, which indicated effectiveness of the model.Hypertension is a serious threat to human health and food-derived ACE inhibitory peptides can regulate high blood pressure without side effects. In this study, incubation conditions of fermented goat milk with


Export of warm and salty waters from the Caribbean to the North Atlantic is an essential component of the Atlantic Meridional Overturning Circulation (AMOC). However, there was also an active AMOC during the Miocene, despite evidence for an open Central American Seaway (CAS) that would have allowed low-salinity Pacific waters to enter the Caribbean. To address this apparent contradiction and to constrain the timing of CAS closure we present the first continuous Nd isotope record of intermediate waters in the Florida Strait over the past 12.5 million years. Our results indicate that there was no direct intermediate water mass export from the Caribbean to the Florida Strait between 11.5 and 9.5 Ma, at the same time as a strengthened AMOC. After 9 Ma a strong AMOC was maintained due to a major step in CAS closure and the consequent cessation of low-salinity Pacific waters entering the Caribbean. During the Miocene, the Central American seaway was not closed, allowing low-salinity Pacific water to potentially weaken the Atlantic circulation. A new, continuous Nd isotope record shows that there was no direct intermediate water mass export from the Caribbean to the Florida Strait and thus, the Atlantic circulation could strengthen. Most model experiments suggest that an open Central American Seaway (CAS) allowed low-salinity waters from the equatorial Pacific to enter the Atlantic at mid-latitudes resulting in an overall weaker AMOC (e.g.6), and a possible link between CAS closure and changes in North Atlantic Deep Water production has been widely discussed (e.g.10). The timing of CAS closure is thus of crucial importance but available reconstructions are controversial, with much of the controversy hinging on the definition of closure11. On the basis of geochemical and geochronological data and paleomagnetic reconstructions, recent studies have argued that the CAS essentially disappeared completely as early as 15 Ma14, although allowing that transient and shallow seaways may have persisted until 10 Ma14. This, however, contradicts biostratigraphic evidence for a gradual shoaling from 15 Ma onwards, with the seaway still 1000-m deep at 12 Ma15. The appearance of a significant and permanent surface water salinity difference between the eastern equatorial Pacific and the Caribbean after ~4.7 to 4 Ma20 points towards a later CAS closure, although changes in sea-surface properties could alternatively be explained by a switch from predominantly El Niño-like to predominantly La Niña-like conditions22. What constitutes ‘closure’ is also under debate where the fossil record is concerned. If shallow seaways with sufficiently high currents, similar to the Indonesian throughflow23, were present in the Pliocene CAS, this may have prevented migration of species between North and South America20 and could explain the divergence of marine molecular DNA on either side of the CAS after 3 Ma20 and the fossil evidence for the Great American Biotic Interchange24 from 2.6 Ma onwards25. If the currents in the CAS were not strong, or if the seaways were transient, then this could explain why a recent study combining fossil and molecular data found waves of terrestrial organism dispersal at ~20 and 6 Ma and divergence of Atlantic and Pacific marine organisms at ~23 and ~7 Ma26.Of the 30 ± 1.5 Sverdrup . The longest record of the Gulf Stream εNd evolution from 850 m water depth in the northwest Atlantic extends back to 8.5 Ma and shows a shift towards less radiogenic compositions between 8.5 and 5 Ma, which was interpreted as a reduction in the advection of intermediate-depth Pacific water to the western North Atlantic28, a conclusion supported by model results29. For deep waters, sedimentary εNd records from the Caribbean extending back to 18 Ma show that the irreversible shift away from Pacific-like compositions already occurred earlier, at 10.7 Ma10. However, in apparent contrast to most model predictions (ref. 5 and references therein), episodes of increased Pacific through-flow in the Middle to Late Miocene Caribbean (between ~12.5 and 10 Ma) were shown to have coincided with increased AMOC strength10.In the light of the ambiguous evidence for the timing of CAS closure, it is pertinent to examine the progressive restriction of water mass exchange via the CAS in more detail and to compare these results with model predictions of the consequences of variations in Pacific water export to the Caribbean and the Atlantic. The radiogenic Nd isotope composition of past seawater has been used extensively to reconstruct changes in the mixing and provenance of water masses (cf. ref. 32. We present a continuous new 12.5 Myr seawater εNd record obtained from sedimentary foraminifera coatings of Ocean Drilling Program (ODP) Site 1006 (658 m water depth) located on the leeward slope of the Great Bahama Bank (GBB)  values of up to −4.6 prior to 11.5 Ma dropping sharply to −8.1 at 10.8 Ma  values then became slightly more radiogenic again between 9.5 and 8 Ma (−6.6 to 7.3 εNd(t)) before decreasing to −8.8 εNd(t) by 6.8 Ma. Thereafter, the εNd signatures at Site 1006 fluctuated between −8.4 and −6.2 until 3.1 Ma to finally become less radiogenic, approaching core-top values of ~−9 to −10 from 0.85 Ma onwards. The εNd(t) signature of the detrital fraction at Site 1006, with the exception of a single sample at 7.5 Ma, varied between −5.6 and −8.5 from 12.5 to 4 Ma and became less radiogenic thereafter (−8.6 to −10.7 εNd(t))  records of Site 100034 and now allow direct comparison of the long-term changes at the two intermediate-depth sites  values reaching −2.6 to −3.1 between 13.6 and 12.1 Ma  decrease to −7.8 between 12.1 Ma and 8.4 Ma and a slight increase to −6.6 between 7.3 and 6.8 Ma. Unlike the Site 1006 record, the detrital fraction of Site 1000 sediments shows a distinct step-change from εNd(t) values between −2.2 and −7.6 prior to 9.4 Ma to between −8.3 and −10.5 after 8.4 Ma  at Sites 1006 and 1000, as well as at deep Caribbean Sites 998 and 99910, was within a range from −4.6 to −2, reflecting a strong Pacific throughflow into the western North Atlantic  signal at Site 1006 compared to Sites 998, 999 and 1000 is consistent with decreasing εNd signatures with increasing distance from the CAS, given that the Pacific signal was continuously mixed with Atlantic waters with a less radiogenic εNd signature36. Likewise, any particulate material from the Pacific transported across the CAS would more likely have been deposited in the vicinity of Site 1000 than at Site 1006, which explains why the detrital fraction at Site 1000 was on average ~2.5 εNd units more radiogenic than at Site 1006 prior to 9 Ma  at Caribbean Site 1000 became progressively less radiogenic by more than 3 εNd units to reach −7.8 by 8.4 Ma. This decrease is similar to the overall trend at two deep Caribbean Sites, which together with increasing sediment accumulation rates was attributed to a decrease in the inflow of more radiogenic and corrosive Pacific waters entering the basin via the closing CAS10. Our new data show that the composition of the intermediate-depth Caribbean was strongly impacted by CAS closure in the Late Miocene, as reflected by a marked drop to less radiogenic detrital εNd signatures after 9 Ma. The εNd(t) and 87Sr/86Sr signatures of the detrital material . However, Newkirk and Martin10 found apparently contradicting evidence that the interval of high Pacific water throughflow into the deep Caribbean between ca. 12.4 and 9.5 Ma corresponded to an increase in the production of NCW, as estimated from δ13C gradients between the Atlantic and the Pacific31. Lear et al.32 also found evidence for a proto-NADW between 12.5 and 10.5 Ma on the basis of cooler benthic Mg/Ca temperatures and higher δ13C signatures. Our new record from Site 1006 now shows that this interval of high NCW production32 ) and hence the strength of the AMOC  as a result of admixture of waters from the western North Atlantic subtropical gyre47. Similar Nd isotope compositions in the Florida Strait33 and the Atlantic gyre47 preclude any calculation of the exact contributions from each source at Site 1006 but low flow speeds in the deep Santaren Channel45 would allow for mixing between the two. On the Pacific side of the Isthmus of Panama, the εNd signatures of intermediate-depth eastern equatorial Pacific water masses are much more radiogenic (−4 to −1.6)48.ODP Site 1000 is today bathed by diluted Antarctic Intermediate Water (AAIW)50.The age models for Site 1006 and for the Miocene part of Site 1000 are based on published biostratigraphySediment samples from ODP Sites 1000A and 1006A were obtained from the IODP Core Repository in Bremen. A further seven core-top samples from the GBB area were obtained from the Seafloor Samples Laboratory of the Woods Hole Oceanographic Institution  from the >350-μm size fraction for the extraction of the bottom water Nd isotope composition from the ferromanganese coatings of the uncleaned foraminifera51. The shells were crushed between two glass plates to open the chambers, and were then ultrasonicated with alternate rinses of distilled (MQ) water and ethanol to remove any detrital particles. A final visual check for removal of particles was performed under the microscope before dissolution of the foraminifera samples in 0.5 M HNO3.Approximately 35 mg of mixed planktonic foraminifera were picked (mostly 53. Samples were rinsed with distilled (MQ) water, followed by a decarbonation procedure using an acetic acid/sodium acetate buffer (with a pH around 4). A hydroxylamine hydrochloride solution was used as a leaching agent for removing the Fe–Mn oxyhydroxide fraction, and was buffered with acetic acid/sodium hydroxide to reach the pH value of 4. This initial leach solution was removed and an additional 12 h of leaching with newly added leaching solution was carried out to completely remove any authigenic coatings. After a triple rinse with MQ water and oven drying, an aliquot of 0.05 g of the remaining detrital fraction underwent total dissolution via hotplate digestion using a mixture of HNO3, HF and HCl. Hydrogen peroxide and HClO4 were included in the procedure for the removal of organic material.To extract the detrital fraction, the 2.5 g powdered aliquots were leached following the procedures fully described in refs. 54 to collect rare earth elements (REEs) and, in the case of the detrital samples, the Sr cut also was collected. Thereafter, Nd was separated from the other REEs 55 and Sr was separated from Rb and other interfering elements 56.Further separation of Nd and Sr and purification of the sample solutions was carried out ion chromatographically. All samples were put through cation exchange columns 143Nd/144Nd results were mass bias corrected to a 146Nd/144Nd ratio of 0.7219 and normalized to the accepted JNdi-1 standard 143Nd/144Nd value of 0.512115 (ref. 57). For downcore samples, εNd(t) values were calculated by correcting the measured εNd(0) for ingrowth of 143Nd using an Sm/Nd ratio of 0.139 for uncleaned foraminiferal calcite58 and 0.109 for detrital material, based on the average upper crustal ratio59. The external reproducibility (2σ) was between 0.21 and 0.40 εNd units for the detrital samples. The external reproducibility (2σ) for the measurements of the foraminifera samples was higher (0.98 εNd units) due to smaller sample sizes that required a time resolved measurement method.Isotope ratios were measured on a Nu Instruments Multiple Collector Inductively Coupled Plasma Mass Spectrometer (MC-ICPMS) at GEOMAR. The 86Sr/88Sr ratio of 0.1194 and normalized to the widely accepted 87Sr/86Sr value of 0.710245 for the NIST SRM987 standard. The 87Sr/86Sr results were corrected for ingrowth of 87Sr using an 87Rb/87Sr ratio of bulk earth of 0.09 (ref. 60). The external reproducibility (2σ) was between 0.000019 and 0.000022.After correction for Kr and Rb interferences, Sr results were mass bias corrected to an Total procedural blanks for Nd chemistry were ≤360 pg and for Sr chemistry were ≤4600 pg. In both cases, these blanks were below 1% of the total sample size and therefore considered insignificant.Supplementary InformationPeer Review File


During ice navigation, blade measurements of ice-induced moments on ship propellers, are challenged by the harsh operating environment. To overcome this problem, shaft line measurements are performed inboard, and the required propeller loads are subsequently estimated using a dynamic model and the solution of an inverse problem. The inverse problem is mathematically ill-posed and requires the determination of the ice-induced moment on the propeller blades from shaft line measurements. Full-scale torsional response data is presented as calculated from indirect strain measurements on the shaft line of a polar supply and research vessel. The vessel operated on a 68-day voyage between Cape Town and Antarctica and spent almost 11 days in sea ice with observed concentrations above 90% and a maximum thickness of 3 m. Data for five ice-induced load cases are presented, including the shaft torque from indirect measurements and the estimated ice-induced moment, which is obtained by solving an ill-posed inverse problem. The ice-induced moments on the propeller are obtained by approximating the drive-train as a viscously damped, elastic lumped mass model. The ice-induced moment is then determined through existing approaches to solving the ill-conditioned inverse problem. The lumped mass model is presented along with algorithms to solve the inverse problem, including truncated singular value decomposition, truncated generalized singular value decomposition and Tikhonov׳s method. The resulting time series data for the inversely calculated ice-induced moments is published to provide industry with load cases for ice-going propulsion design. Specifications TableValue of the Data•Shaft-line torque data are presented for operational incidences of propeller ice impacts on a polar supply and research vessel on a voyage between Cape Town and Antarctica. The data included the maximum ice-impacts as measured on the shaft-line, although this does not necessarily imply that these load cases are also the maximum ice-induced moments on the propeller blade.•Time histories of the induced propeller torque are published as determined through inverse moment calculations, by using a viscously damped, elastic, lumped mass structural model.•This data provides industry with operational load cases for ice-going propulsion design.•The co-publication of time histories from inversely calculated shaft moments enables the validation and further development of methodologies for inverse moment estimation.1To determine the loading contribution of ice impact, the hydrodynamic torque was subtracted from the measured internal torque and the direction of the moment inverted to obtain a positive external ice-induced moment on the propeller. It was further evaluated if the estimated ice moment could again be inverted to match the measured shaft torque value. This inverted internal torque was obtained by determining the relevant external moment through the Tikhonov method and using this result as an input to the dynamic model to obtain the internal torque (by solving the forward problem).The time series data of five propeller ice impact cases are published here as shown in 22.1The SAA II, depicted in Shaft-line measurements were performed during the 2015/2016 Antarctic relief voyage of the SAA II as presented by the GPS track in 2.2Visual observations of ice conditions were performed from the bridge of the vessel when operating in ice as comprehensively documented by Suominen et al. The ice thickness was estimated by comparing the thickness of the cross-section of upturning ice debris which was scaled with the aid of a yard stick. The yard stick was suspended overboard from the main deck and was marked with 10 cm wide black and white markings to calibrate the estimations of observers, see The concentration of the ice field was estimated from inboard observations from conditions experienced in the close vicinity of the ship. As the crew preferably navigated in open water instead of ice, the ship followed open water leads in the ice whenever possible. In this case, the concentration was marked as zero, although floes of ice could be seen. The range from 0 to 100% was divided into categories with 10% increments, i.e. 0–10%, 10–20%, for observations of ice concentration. Here, zero denotes open water and 100% indicates complete ice cover. Ice floes were categorized in terms of diameter in categories which included <20 m, 20–100 m, 100–500 m, 500–2000m, 2–5 km, >5 km. The classes were selected based on the egg code The total voyage lasted 68 days, of which 10.7 days were spent navigating in ice, 40 days navigating in open water and 17.5 days stationary. The pie chart in 2.3.Strain gauges were installed on the port side intermediate shaft line, 25.9 m from the center of gravity of the propeller , to deteThe torque in the shaft, Here, 2.4Ice-induced moments on the propeller are to be determined from indirect shaft line measurements. This is achieved through a two-step process. Firstly, a forward problem is solved whereby the dynamic model is subjected to a step impulse moment at the propeller. The impulse response function between the externally applied ice moment on the propeller and the internal torque response in the shaft line is thereby determined at the measurement location. Secondly, an inverse problem is solved to determine externally applied propeller moments from the measured shaft line torque and ill-posed inverted impulse response.A simplified dynamic model of the torsional dynamic response of the SAA II was obtained by using a lumped mass model documented by Rolls-Royce Here, ,, …,,, …,A diagram of the lumped-mass model for the SAA II shaft line is shown in In order to determine the ice-induced torque at the propeller, rotational degrees of freedom were defined along the longitudinal shaft axis (x-axis). The model comprised two types of elements, namely inertia elements and shaft elements. The respective elements each contained two nodes as shown in with Direct integration methods could be used to obtain approximate solutions of dynamic systems The dynamic model was solved by first defining an integration formula for the angular velocity, 2.4.1The principle of superposition Eq. Here, The complication with the discretization of inverse problems is that this leads to an ill-conditioned coefficient matrix for the system of linear equations, which require regularization methods to obtain stable solutions To this end three inverse methods have been investigated to perform inverse moment determination in an impact loading situation of the dynamic shaft line structure. In keeping with the approach of Ikonen et al. TSVD is a common method used to regularize ill-posed systems. The SVD of Here, Truncated Generalized Singular Value Decomposition (TGSVD) is a more sophisticated method whereby further information about the desired solution can be incorporated to stabilize the problem The system can be represented by the real matrix pair (Here, Another widely used regularization method is Tikhonov׳s regularization method, which involves the solution of a least squares problem. This method is convenient for problems in which both the coefficient matrix and the required solution can only be determined approximately λ is a positive constant referred to as the regularization parameter. The required solution for the ice moment vector, Here, 2.4.2The solution of ill-posed inverse problems using TSVD and the Tikhonov methods, require the determination of the respective regularization parameters, Synthesized moment impulses were created, with the requirement that it be representative of a real ice induced loading moment. A linear impulse of The dynamic model applies the change in external moment, therefore the first time derivative of the known moments need to be determined. This was done with a time step of In order to apply inverse methods, the regularization parameters, This plot is only applicable to the TGSVD and Tikhonov methods as SVD does not implement the L-matrix. The zero, first and second order regularization matrix L was evaluated for the current model and it was determined that the first order regularization matrix provided the best results for all three inverse methods. The optimum number of non-zero eliminated singular values for GSVD was determined to be 2.4.3Matlab algorithms are published with this data to load the load case data and perform the required inverse calculations. The procedure followed by the Matlab algorithm, InverseMethod.m is outlined in


Nature Communications 10.1038/s41467-019-09987-0, published online 26 April 2019.Addendum to: The authors have become aware that ‘BTR pseudo-site 2’ inadvertently contains two incorrect nucleotides and does not match the sequence of the pseudo-site in the human genome. The data points corresponding to this construct in Fig.


Xenograpsus testudinatus was firstly discovered in 2000 at the hydrothermal vent field off the coast of Kueishan Island. The present study attempts to understand the adaptive reproduction of this crab living in an extreme environment by examining its spatial and temporal distribution and isotopic signatures. The seasonal variation of the female-male ratio suggests that ovigerous females may migrate from beneath the vent orifice to the vent-periphery region to release their larvae to avoid the larvae contacting high toxic plumes, and then returns to the vent orifice habitat. We used variation of the isotopic crab signatures as indicators for this unique female migration. Our results showed that this vent crab evolved an adaptive modulation of reproductive behavior to successfully survive and propagate in an oceanic shallow hydrothermal vent field.The vent crab  Hydrothermal vents exist in locations ranging from coastal ridges to the abyss. Light is most likely absent at depths beyond 200 meters, and biological communities are dominated by numerous symbiotrophic forms, sustained by chemosynthesis using sulfide compounds as their primary energy source , 2. In cXenograpsus testudinatus is currently the only crustacean species known to live in the shallow hydrothermal vent field of Kueishan Island. This vent crab is believed to have lived in the area for a long time, but was only discovered in 2000, and appears to have adapted to such extreme local environment. Jeng et al. [X. testudinatus living in the vent field likely fed on zooplanktons that were killed by the vent plumes and then settled on the sea-bed like ‘snow.’ Although some limited reports have examined the diet, metabolic and behavioral adaptions of X. testudinatus in the shallow hydrothermal vents [Kueishan Island, a young active volcanic island, is located approximately 10 km off the northeastern coast of Taiwan. To date, the shallow hydrothermal vents found near Kueishan Island have predominantly been found on its eastern side. g et al.  proposedal vents , 6, 7 reBythograea thermydron has only been observed in the vent periphery and not in the vent orifice [X. testudinatus. This study attempts to elucidate crab adaptation and reproductive biology in this extreme environment by examining crab ovigerous rates, seasonal shifts in sex ratio, isotopic signatures and possible food sources.In deep hydrothermal vents, the ovigerous female of the vent crab  orifice . TherefoFig 1). The crab Xenograpsus testudinatus and linked organisms were caught in April and July 2010. Crabs were not caught at site ‘B’ in the April sample because of extremely bad weather. Site ‘A’, with a vent orifice, is located on the eastern side of Kueishan Island. Site ‘B’ is also a vent location, emitting fluids likely from crevices, but located some distance from site ‘A.' Meanwhile, site ‘C’ is located on the edge of the vent field where X. testudinatus can be found with other organisms. Finally, site ‘D’ is a coral reef area located in the north of Kueishan Island where no vent crabs can be found. Additionally, a home-made sediment trap  was deployed in both vent field and coral reef sites for eight days during September 2011 to collect settling materials.In this study we analyzed seawater, sediment, and biological samples collected by researchers through three independent samplings from shallow water hydrothermal vents, conducted in April 2010, July 2010, and July 2011 at four sites  and nitrogen (TN), and total inorganic carbon (TIC) and inorganic sulfur (TS) in sediment and trap samples were measured according to the previous methods of Hung et al. [13C and δ15N) data measured from X. testudinatus and other organisms caught in July 2010 according to the analytical method of Liu et al. [During crab and sediment sampling expeditions, temperature, dissolved oxygen (DO), pH, and total dissolved sulfide (TDS) in seawater were measured g et al. , 10, 11.u et al. . The col13C and δ15N in X. testudinatus, ovigerous rate and elemental composition in sediment from each sampling site were statistically analyzed using ANOVA and then compared using the Fisher LSD test. The student t-test was used to compare the differences in elemental composition between the trap samples collected from the vent field and coral reef field of female and ovigerous females.The data of δFig 2) demonstrated that the crabs from beneath the vent orifice (site ‘A’) have total female and ovigerous female ratios of 22% and 0% in April, respectively. The total female and ovigerous female ratios increased to 65% and 2.04% in July 2010. In contrast, the total female and ovigerous female ratios at site ‘C’ in April were 72% and 46.15%, respectively. Both ratios decreased to 48% and 28.87%, respectively, in July 2010. The results indicate that spring is the main reproductive season of X. testudinatus, and the mature female crab may exhibit unique seasonal-migration behavior during this period. The isotopic signatures of collected female and male crabs also indicated the unique migratory behavior of female crabs .Comparing the female ratio and ovigerous female ratio of the vent crabs at the three collection sites during different seasons  suggested that the food web structures contained primary and secondary consumers. The isotopic signatures of male crabs collected during July 2010 were categorized into three different groups , corresponding to their different habitats  . The isotopic signatures in group ‘A’ differed significantly from those in groups ‘B’ and ‘C’ . However, the signatures in group ‘B‘ resembled those in group ‘C’.In this study, δ-1). The seawater samples collected from the bottom of site ‘A’ (the bottom of the vent chimney) showed values similar to the ambient ocean waters: temperature (27°C) and DO (179.5 μM). These waters show a slightly lower than normal pH (6.38) and TDS (5.49 mg L-1). Such distributions do not differ significantly compared to those from the non-vent region , except in pH and TDS (Table 1). Comparing the composition of the settling materials collected from vent and coral reef fields revealed significantly higher concentrations of total organic carbon (TOC) and total sulfur (TS) in the vent field  than the coral reef field  (Table 2). The low C/N ratios detected in the settling materials from the vent field may also imply that they have a relatively high zooplankton content, which was confirmed by examination using a microscope .The vent fluid collected at site ‘A’ showed high temperature (106°C), low pH (2.48), low DO (47.3 μM), and high TDS  suggested that the food web structures consisted of primary producers and secondary consumers. For the female crab, the isotopic signatures of specimens collected from site ‘A’ were categorized into groups ‘B’ and ‘C’ but not group ‘A’. Apparently, the non-ovigerous female individuals at site ‘A‘ were recent migrants from sites ‘B’ or ‘C’ in July 2010. This adaptive behavior may be related to the physical, chemical and biological characteristics of their habitats.Analysis of stable isotopic signatures of δstaceans . δ13C an-1), this fluid is potentially toxic and fatal for crabs. However, the seawater collected from the bottom of site ‘A’ (the bottom of the vent chimney) showed values similar to the ambient ocean waters: temperature (27°C) and DO (179.5 M); these waters show a slightly lower than normal pH (6.38) and TDS (5.49 mg L-1). Such distributions do not differ significantly compared to those from the non-vent region , except in pH and TDS (Table 1). This way adult crabs which stay at the bottom avoid direct contact with hot and toxic fluids.Since the vent fluid collected at site ‘A’ showed high temperature (106°C), low pH (2.48), low DO (47.3 M), and high TDS (26.5 mg LS1 Video) will drift with the upwelled vent fluid and may suffer immediate injury or death if they were released by an ovigerous crab at site ‘A’. Consequently, the mature female crabs migrate to the vent-periphery region (site ‘B’ or ‘C’), about 100–200 meters horizontally away from the vent opening to release their larvae in the ambient environment there, which is more conducive to the larval development.Larvae which exhibit planktotrophic behavior . This indicated that these materials largely resulted from the resuspension of bottom sediments. In conclusion, this study demonstrated that the vent crab X. testudinatus has developed a unique reproductive migratory behavior as an adaptation to the shallow hydrothermal vent field. The mature female crabs migrate to the vent-periphery to release their offspring to prevent injury and toxic effects from hot and toxic vent fluids. After larval release they migrate back to the vent field, an area enriched with zooplankton food and with minimal competition or predation from other organisms, indicating a behavioral reproductive adaptation to the vent system. The present study supports the concept of marine hydrothermal vents as peculiar oceanic environments as proposed by Dahms et al. [The reason for female crabs migrating back to their original habitat, the vent field after releasing their larvae is an interesting issue. Lack of competing organisms or predators and rich food sources in the vent field are possible explanations. The settling materials in the coral reef field were characterized by less zooplankton but more macro-algal fragments and mineral particles  inside to collect the sink materials. Right: The picture of the trap (DOCX)Click here for additional data file.S2 FigLeft: vent field, with many zooplankton ; Right: coral reef field, less zooplankton but have more macro algae fragments than vent field samples.(DOCX)Click here for additional data file.S1 Video(AVI)Click here for additional data file.


Escherichia coli is required to prevent immediate re-initiation of chromosome replication from oriC. The SeqA protein is phosphorylated at the serine-36 (Ser36) residue by the HipA kinase. The role of phosphorylation was addressed by mutating the Ser36 residue to alanine, which cannot be phosphorylated and to aspartic acid, which mimics a phosphorylated serine residue. Both mutant strains were similar to wild-type with respect to origin concentration and initiation synchrony. The minimal time between successive initiations was also unchanged. We therefore suggest that SeqA phosphorylation at the Ser36 residue is silent, at least with respect to SeqA's role in replication initiation.The SeqA protein of  Escherichia coli the DnaA initiator protein binds ATP and ADP with equal affinity  and a C-terminal DNA-binding domain , which are joined by a flexible linker -based quantitative phosphoproteomic approach combined with high-resolution mass spectrometry identified residue serine-36 (Ser36) in SeqA as a direct phosphorylation target for the kinase activity of the high persister protein A, HipA  or aspartic acid (S36D). The S36A mutation impairs Ser36 phosphorylation, whereas the S36D mutation mimics the conformation of Ser36 phosphorylated SeqA .Cells were grown in AB minimal medium , base substitutions were made in the codon for Ser36  to Ala(A), and 5′-TCC-3′ to 5′-GAC-3′; Ser(S) to Asp(D), respectively) using splicing by overlap extension (SOEing) polymerase chain reaction (PCR)  The lower region of the seqA gene, spanning residues 27–181, was amplified using primers “SeqA_down_bw_XmaI” and either “SeqA_pos36_SA_fw” or “SeqA_pos36_SD_fw”. 2) The upper region of the seqA gene was amplified using primers “SeqA_up_fw_SacI” and “SeqA_intern_bw” that generates a fragment with an overlap of 21 bp with the seqA downstream PCR product. A secondary amplification was performed using equimolar ratios of the two PCR products as template, and the oligonucleotides, SeqA_down_bw_XmaI, and SeqA_up_fw_SacI, as primers. The resulting PCR fragments were digested with XmaI and SacI, and cloned into the same sites of the 3.9 kb suicide vector, pRUC1437, a derivative of pSW29T  from MG1655 lacIZYA::cat cells carrying either the seqAS36A or seqAS36D chromosomal genes (see above), respectively, using DNA oligonucleotides, SeqA_up_fw_EcoRI and SeqA_down_bw_HindIII, as primers. The resultant PCR fragments were digested with EcoRI and HindIII and inserted downstream the IPTG inducible lacPA1−04/03 promoter  were treated with rifampicin  and cephalexin  to inhibit initiation of DNA replication and cell division, respectively  were harvested. Proteins were separated by SDS-PAGE and SeqA protein detected by Western blot using rabbit antiserum raised against SeqA protein , Vibrio cholerae (accession: AOY47782.1), Pasteurella multocida PM70 (accession: AAK02440.1), Haemophilus influenzae Rd (accession: NP_438362.1), Yersinia enterocolitica (accession: CNB62546.1), Serratia marcescens (accession: KFL03527.1), Actinobacillus pleuropneumoniae (accession: SQF64393.1), and Glaesserella parasuis (accession: STO80764.1).Multiple alignment analysis of SeqA amino acids sequences was performed in the MEGA version 7.0.26 software . The SeqAS36D mimics the conformation of Ser36 phosphorylated SeqA . The resulting protein, SeqAS36A, is phosphorylation impaired at position 36  = 0.96]. The structural differences caused by changing the Ser36 residue seem to affect only the flexible linker region between SeqA-N and SeqA-C , phospho-mimetic (S36D) and wild-type SeqA proteins.seqA, seqAS36A, and seqAS36D genes from the IPTG-inducible lacPA1/04−03 promoter in SeqA deficient cells. Exponentially growing cells were induced with 1 mM IPTG at time 0 min (T = 0 min). Immunoblot analysis of cells sampled at 120 min following the addition of IPTG indicated that all SeqA proteins were expressed to comparable levels corresponding to an ~12- to 14-fold increase in SeqA level relative to wild-type cells , but not at 42°C (non-permissive temperature) due to a reversible defect in nucleotide binding (Carr and Kaguni, seqAS36A and seqAS36D cells carrying the dnaA46 allele were grown exponentially at 30°C. The average number of origins per cell for all three strains was close to 2 (Figures seqA mutant cells (Figures We introduced the  Figures  and the  Figures . When ce Figures . Upon a  Figures . This 20 Figures , and it  Figures . SeqA de Figures .Vibrio cholerae, Pasteurella multocida PM70, Haemophilus influenzae Rd, Yersinia enterocolitica, Serratia marcescens, Actinobacillus pleuropneumoniae and Glaesserella parasuis with that of E. coli K12. All of these bacteria are known to carry hipBA genes. We looked for conservation of Ser36 along with the two flanking amino acids Phe35 and Ala37 (Table We aligned SeqA amino acid sequences from the 37 Table . None of37 Table , were co37 Table  we also 37 Table . This maE. coli cells, and was revealed as a direct phosphorylation target of HipA in vitro. When the hipA gene was expressed from a p15A based plasmid, the fraction of wild-type SeqA found to be phosphorylated at residue Ser36 was ~7% following 95 min induction (Semanjski et al., Recently, it was shown that residue Ser36 in the SeqA protein is a target for phosphorylation by the serine-threonine kinase, HipA (Semanjski et al., S36A, and SeqAS36D proteins that indicated the mutations to cause minor structural changes to the linker region only, leaving the N- and C-terminal domains unaffected. This might explain our observations that function and activity of the SeqA mutant proteins seemed unaffected by the Ser36 mutations with respect to replication initiation control.The Ser36 residue is located in the flexible linker between the N-terminal oligomerization domain and the C-terminal DNA binding domain (Chung et al., S36D protein may deviate in activity from the phosphorylated wild-type SeqA protein.Although we have assumed that substituting a serine residue with a negatively charged amino acid, such as aspartic acid, imparts the negative charge associated with serine phosphorylation, caution should be taken as this is not always the case. The phospho-mimetic proteins may fail to recapitulate the true steric and charge-based nature of phosphorylation (Paleologou et al., hipBA carrying bacterial species compared to highly conserved amino acids crucial for oligomerization and DNA binding activity.However, because removal of the HipA kinase in wild-type cells revealed no replication phenotype, we find it unlikely that HipA-mediated Ser36 phosphorylation affects the activity of SeqA, at least with respect to its function in replication initiation, and at least under the conditions provided in this study. SeqA phosphorylation may therefore be an example of a silent phosphorylation. This has previously been observed for pepsin and ovalbumin, where serine phosphorylation did not affect protein activity, and the function of the phosphate group remained unknown (Johnson and Barford, LR, EG, and AL-O planned the experiments. LR, BK, and LK performed the experiments. LR, BK, LK, and AL-O analyzed data. LR, BK, and AL-O wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T Phosphorylation plays a ubiquitous and essential role in regulating almost all biological functions. The level of oligonucleotide and peptide phosphorylation is well modulated by both kinases and phosphatases –5. Althoα-hemolysin) exhibits less sensitivity for directly identifying the phosphorylation states of oligonucleotides through the reading of ionic current. Therefore, an efficient nanopore sensing interface with robust interactions with the phosphate groups of oligonucleotides/peptides is urgently demanded for directly distinguishing the phosphorylation from the native substrates. An aerolysin nanopore owns an extremely strong sensing interface to interact with charged oligonucleotides [β-barrel without the needs of motor proteins or labels [4 for the model T4 oligonucleotide kinase (PNK) to achieve the proof-of-concept experiments which are aimed at label-free evaluation of phosphorylation/dephosphorylation-related enzyme activity with nanopore methods. PNK plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and the repair of DNA during strand interruption [A single biological membrane protein-based single-biomolecule interface provides a single-molecule nanopore platform for sensitive detection of DNA/RNA –22, peptleotides –40 and pleotides , 25, whirruption , 42. Ourrruption , 43. The4 (5′-AAAA-3′) and poly(dA)5 (5′-AAAAA-3′) were chosen as the model oligonucleotide substrates. First, the poly(dA)4 and poly(dA)5 were added into the cap side of the aerolysin nanopore. The applied potential was set at +120 mV while the cap side was defined as a virtual ground. Both poly(dA)4 and poly(dA)5 without phosphorylation illustrate one prominent translocation population in the scatter plots of the duration and residual current ratio (I/I0) n (3 ≤ n ≤ 10) produces the sharp distribution of blockage current with duration time as long as tens of milliseconds [5 owns one nucleotide longer than poly(dA)4, it leads to a larger current blockage amplitude with a statistical I/I0 of 0.39. After the phosphorylation at 3′ end, the poly(dA)4-3′-P hardly produces the blockages longer than 0.5 ms 5′-3′-P. The detail mechaniusms of the phosphorylation-driven acceleration are currently under studying in our group.As shown in iseconds . Since tn 0.5 ms , left. Shan 1 ms , left. S3 with three negatively charged glutamic acids. I/I0 = 0.53 and statistical fitted duration of 0.25 ms. This result is consistent with our previous study [3, which enhances the electrophoresis force for a fast peptide translocation. Therefore, the rapid translocation of phosphorylated (EYQ)3 hardly shows any blockages due to the bandwidth limitation of the amplifier. (3 and phosphorylated (EYQ)3 demonstrate that the aerolysin nanopore could be extended to the evaluation of the phosphorylation for the negatively charged peptide. Then, we further study the aerolysin sensing ability of positively charged peptide with and without phosphorylation. Under the applied potential of +120 mV, the positively charged peptide of LRRASLG hardly enters into the aerolysin nanopore, resulting in few blockages , the blockage frequency in the main distribution would be decreased accordingly. As to the positively charged peptide, the phosphorylation allows it to enter and then interact with the β-barrel of the aerolysin. Therefore, the phosphorylation of positively charged peptide “turns on” the characteristic blockages in the main distribution. Note that the difference of the duration time and frequency depends mainly on the initial net charges and the length of the native oligonucleotide/peptide. Moreover, previous studies showed [n (3 ≤ n ≤ 10) could reach to as low as 1.0 × 10−13 M while that for the short peptide is expected to a nanomolar level. The high capture rate of both oligonucleotides and peptide ensures the low limit of detection for the phosphorylation sensing without amplification methods. Therefore, the aerolysin nanopore could achieve direct and label-free discrimination of single-molecule phosphorylation of both oligonucleotides and peptides via monitoring the frequency and duration responses from the characteristic blockages.Unlike the oligonucleotide carriers of uniform negative charges, the peptides always bear nonuniform negative and positive charges. Therefore, the nanopore phosphorylation sensors for peptides not only should be sensitive to the phosphorylation of negatively charged peptide but also are supposed to generate the distinguishable blockage differences for the phosphorylation of positively charged peptide. To demonstrate the sensing ability of the aerolysin nanopore, we first used a peptide sequence of (EYQ)plifier. , left. Tlockages . After ps showed , 25, 44 2+, PNK can remove the 3′phospotase group of poly(dA)4-3′-P [μL reaction solution containing 100 μM poly(dA)4-3′-P and 10 mM MgCl2 was incubated with a certain concentration of PNK at 37°C. The reaction was quenched by heating to 65°C for 20 min to denature the enzyme after incubation for 1 h. Full experimental details can be found in Supplementary Information. The reaction solution was premixed with 900 μL electrolyte solution and then added into the cap side chamber. After the insertion of the aerolysin nanopore to the lipid bilayer, the analyzed solution containing 100 μM poly(dA)4-3′-P without PNK hardly induces the typical current blockages with a long duration at +120 mV 4-3′-P , 45. The +120 mV  while th +120 mV b–d. The  and 0.6 . The enlμL were successively added into the cap side of the aerolysin nanopore. Here, we defined the frequency of typical blockage events in the target distribution as fr. When the concentration of PNK increases from 0.01 to 0.1 U/μL, the fr increases from 14.47 ± 0.04 s−1 to 46.18 ± 0.13 s−1. There exists a positive correlation between PNK concentration and fr 4-3′-P and 10 mM MgCl2. Hardly, any typical blockage events were observed before the addition of PNK to the chamber  the strong ability for the sensitive response to the charge states of both DNA and peptide substrates thereby directly regulates the event duration as well as the frequency without exogenous labels, immobilization, and cooperative enzymes. Employing this generalized nanopore approach, one has the potential to characterize the complex regulatory process resulting from both DNA and protein kinases/phosphatases. (2) Few ionic responses to the background  ensure tWe could anticipant that our generalized strategy facilitates the understanding of phosphorylation-involved cellular signaling pathway. Moreover, the diagnostic application would build upon our initial findings with the further improvements of clinical sampling system .