Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome–enzyme complex. Cysteine side chains are reactive sites of protein synthesis and modification. Here, the authors show that tuning palladium chemoselectivity allows for selective removal of several cysteine protection groups, and use their method to synthesize challenging protein analogues. These analogues have been utilized in various biochemical, structural, and functional studies. Much of what has been done in the area of modifications of expressed proteins is based on the chemistry of the thiolate side chain of the Cys residue;5 either by employing direct alkylation/arylation or by transforming it to other functionalities, such as dehydroalanine (DHA)6. The latter approach has expanded the chemistry for modifications and the ability to generate protein activity-based probes9. The chemistry of the Cys side chain has also been critical in both the total chemical synthesis and semisynthesis of proteins11. For example, native chemical ligation (NCL)12, which is the most utilized approach for assembling the desired polypeptide sequence, relies on the presence of N-terminal Cys, β-, γ- and δ-thiolated amino acids1, or a thiol modified auxiliary16. The thiol handle in these derivatives promotes trans-thioesterification with a peptide thioester for the subsequent S-N acyl transfer and formation of an amide peptide bond at the ligation site.Organic chemistry has revolutionized the field of protein science by contributing efficient synthetic tools to prepare various protein analogues, such as proteins labeled with fluorophores, D-amino acids and posttranslational modifications (PTMs)17. The site-specific removal of a particular PG makes it possible to control the direction of synthesis  and/or the modification site. Several reports have demonstrated the usefulness of NCL coupled with orthogonal protection for the synthesis of highly complex analogues20. Examples include the synthesis of branched proteins, such as ubiquitinated proteins;23 proteins containing multiple Cys residues ;25 and site-specific antibody–drug conjugates26. In these cases, orthogonal protection of Cys residues, mainly by using thiazolidine (Thz)27 and acetamidomethyl (Acm)28, is a critical step in obtaining the product in the desired form30. Despite the utility of the current toolbox for Cys protection/deprotection, there are still limitations to these strategies. The need for harsh removal conditions, prolonged reaction times and additional HPLC-purifications steps all limit the application of these approaches to more challenging systems. Therefore, expanding the available toolbox to manipulate Cys PGs in an orthogonal manner would enable the synthesis and study of complex protein targets. We recently reported that palladium complexes can remove multiple Cys PGs within minutes in a fully aqueous medium34, which also could be coupled in situ with NCL conditions to provide excellent yields of the desired products36. Although these conditions have resulted in important advances in chemical protein synthesis34, the utility of these approaches is limited by the lack of chemoselectivity of the palladium complexes when applying more than one PG in the synthesis.When considering the multistep synthesis of different protein analogues, there is an intrinsic requirement for incorporating orthogonal protecting groups (PGs) of the thiolated amino acidst-butyl PG using palladium in a fully aqueous medium and demonstrate its stability under the removal conditions of Thz and Acm. The chemistry reported here is used for the one-pot and site-specific modification of peptides containing multiple Cys residues, as well as for the total chemical synthesis of copper storage protein 1 (CSP-1), which contains 13 Cys residues. We also show the total chemical synthesis of an activity-based probe of ubiquitinated histone H2A at Lys119 to prepare ubiquitinated nucleosome core particle probes (NCP-(H2A-UbDHA)). This probe exhibits reactivity with the Calypso/ASX heterodimer deubiquitinase (DUB) to form a stable covalent nucleosome–enzyme complex.Here we demonstrate palladium chemoselectivity in a fully aqueous medium for on-demand deprotection of Thz and Acm protections. We also show efficient removal of Cys 2 under physiological pH32. Notably, when GSH was used in a 1:1 molar ratio to [PdCl]2, we found that breaking the Thz linkage was more effective compared to using 4-mercaptophenylacetic acid (MPAA) and tris(2-carboxyethyl)phosphine (TCEP) additives31. This prompted us to revisit the Thz and Acm deprotection via palladium, by synthesizing the model peptide, Thz-LYRAGC(Acm)LYRAG (peptide 1), bearing N-terminus Thz and internal Cys protected with Acm. When this peptide was exposed to [PdCl]2 and GSH (1:1) in 6 M Gn·HCl, pH~6.5 at 37 °C, we observed within 45 min the full unmasking of the Thz to the free N-terminal Cys. Surprisingly, the Acm under these conditions was completely stable. Interestingly, upon addition of an extra 10 equiv. of [PdCl]2 to the reaction mixture, the Acm was completely removed within 5 h Cl]2 and GSH  as an additive can accelerate the cleavage of peptides and proteins connected via a Thz linkage using [PdCl]37. With this in mind, we decided to examine the removal of the two PGs in the absence of guanidine. Here we found that when exposing peptide 1 to PdCl2 in water only, we observed reversed and efficient removal of the Acm within 30 min, while the Thz remained completely stable  PG38, which has been used for regioselective disulfide bond formation in peptides and proteins. This PG can be removed under extremely harsh conditions, such as trifluoroacetic acid and 2,2-dithiobis(5-nitropyridine), hydrofluoric acid or by using mercury metal in organic solvents39. We wondered if palladium could assist the removal of the t-butyl in an aqueous buffer. In order to test this, we synthesized the model peptide AC(t-butyl)LYRAGLYRAG (peptide 2) and exposed it to various removal conditions employing different palladium complexes, additives, and temperatures LYRAG. By exposing this peptide to [PdCl]2 and GSH in 6 M Gn·HCl, followed by the addition of an extra equivalent of [PdCl]2, we were pleased to observe the sequential removal of Thz and Acm while keeping the t-butyl intact LYRACLYRCA (peptide 3) for sequential alkylation. To alkylate the free Cys first, we treated peptide 3 with halogenated-acetyl derivative, which was completed within 30 min Cl]2/GSH, this enabled selective Thz removal without affecting the three Phacm tags to provide CSP-1–4. Subsequently, the second ligation with fragment CSP-1-3 was performed to give CSP-1-5. Finally, the three tags were removed using PdCl2 for 1 h to provide the native CSP-1 protein, which showed by circular dichroism (CD) the expected secondary structure  where the ubiquitin carboxyl terminus is covalently linked to the ε-amino group of lysine via an isopeptide bond. In this probe, Gly76 is replaced with DHA to trap the DUB catalytic Cys, as we previously reported for diubiquitin activity-based probes9. Ubiquitination of H2A is a hallmark of heterochromatin, which is dynamically regulated by Calypso/ASX in Drosophila44, and plays an important role in cell cycle control, DNA damage response, and gene regulation45. We anticipate that this type of chemistry could provide useful reagents that would assist in understanding the structural and functional mechanisms of Calypso/ASX DUB in chromatin context.Ubiquitin activity-based probes have found great utility in understanding the function and structure of deubiquitinases46. To enable ubiquitination through Lys119 and latent DHA formation, fragment H2A-1 was prepared bearing also Cys(t-butyl) on Lys119. This Cys, which replaces Gly76 of Ub would serve after H2A polypeptide assembly for the ligation with Ub and DHA formation. Here we envisioned selective removal of the Thz for sequential backbone ligation in the presence of the orthogonally protected Cys. In this synthesis, Ala at positions 86 and 48 in H2A-2 and H2A-3 fragments were temporarily mutated to Cys to enable NCL. With this design in mind, we performed the first ligation between H2A-1 and H2A-2, followed by MgCl2 treatment to enable Thz removal via [PdCl]2. Subsequently, this enabled a second NCL step with fragment H2A-3 to provide H2A(1-129)K119-C(t-butyl). At this stage, we performed a desulfurization step in a one-pot operation without affecting the t-butyl protection to give the full-length H2A-5. The purified intermediate H2A-5 was dissolved in urea or Tris buffer at pH 7.5 and treated with PdCl2 to unmask the Cys(t-butyl) to give H2A(1–129)K119Ub-Cys and was ligated with Ub(1–75)-thioester to furnish the ubiquitinated H2A-6. Subsequently, treatment with the bisamide1,4-dibromobutane47 converted the Cys side chain at the ligation site to DHA to provide the desired H2AK119DHAUb probe  in addition to Ub1-75)-thioester  using standard methods49. Under these conditions, we observed negligible labeling with Calypso/ASX DUB, perhaps suggesting that chemical modification of the DHA moiety may be producing an inactive species. To test this, we then moved to examine the probe stability in the presence of several solution components present during NCP reconstitution, including different buffers (Tris and HEPES), additives (TCEP), and temperatures . To study this, we synthesized a ubiquitinated C-terminal H2A peptide probe (H2A(116-129)K119DHAUb)  probe  probe was incubated for 1 h at 25 °C with Calypso/ASX at concentrations of 3 µM and 6 µM, respectively. The resultant products were analyzed by electrophoretic mobility shift assay (EMSA), SDS-PAGE, and Western blot. Taken together, these results indicate the formation of a unique covalent complex was formed between Calypso/ASX and NCP-UbDHA. The EMSA experiment showed the formation of a band corresponding to nucleosomal DNA migrating at a higher apparent molecular weight than NCP-UbDHA alone. We also observed no dissociation of this complex after serial dilution of the reaction mix supporting the formation of enzyme stably bound to the nucleosome probe , consistent with the formation of Calypso/ASX-H2A-UbDHA complex  to the Calypso/ASX DUB  complex, which has opened up the opportunity for structural studies that would otherwise be very difficult to attempt.In conclusion, we have introduced synthetic approaches to expand the chemical toolbox for synthesizing and modifying peptides and proteins. The reported approaches make possible on-demand palladium-mediated decaging of various Cys PGs to facilitate peptide modification and protein synthesis of complex targets. Remarkably, the selectivity has been achieved by simple variation of the additives and the solvent in the reaction mixture containing commercially available palladium complexes. The palladium tunability that has been achieved here should also initiate other research directions in aqueous-based chemistry of small molecules and trigger mechanistic studies. The synthesis of protein targets, such as the CSP-1 and the activity-based probes based on ubiquitinated H2A, pave the way for answering fundamental questions related to these systems. Of particular interest is the Calypso/ASX-NCP was dissolved at 175 µL, 6 M Gn·HCl, 200 mM Na2HPO4 buffer pH 6.5, and treated with 10 equiv. of [PdCl]2 and GSH (1:1) and incubated at 37 °C for 45 min to observe complete Thz opening. Subsequent addition of extra 10 equiv. of [PdCl]2 to the reaction mixture led to complete removal of the Acm within 5 h.0.5 mg of peptide 1  was dissolved at 175 µL, H2O, and treated with 10 equiv. of PdCl2 and incubated at 37 °C for 30 min to observe complete Acm deprotection. Subsequent addition of [PdCl]2 and GSH (1:1) in 6 M Gn·HCl, 200 mM Na2HPO4 buffer pH 6.5 to the reaction mixture at 37 °C led to complete Thz removal within 45 min.0.5 mg of peptide 1  was dissolved at 500 µL, 6 M Gn·HCl, 200 mM Na2HPO4 buffer pH 8, and treated with 1 equiv. of N-(bromoacetyl)glycine-ethyl ester, which pre-dissolved as a stock solution in MeOH. 100 equiv. of NaI was added to accelerate the alkylation reaction. The reaction was monitored by HPLC-MS, which showed completion within 30 min. Then, the reaction mixture was treated with 10 equiv. of [PdCl]2 and GSH (1:1) and incubated at 37 ° for 45 min to observe complete Thz removal. For the second alkylation, the solution mixture was treated with Cys-PEGA resin for 1 h at 37 °, followed by the addition of 1 equiv. of DTT. Subsequently, 11 equiv. of the N-(bromoacetyl)alanine-methyl ester dissolved in MeOH and 100 equiv. NaI were added and incubated at 37 °. The reaction was completed within 30 min. For Acm removal, 10 equiv. of PdCl2 was added to the reaction mixture and incubated for 30 min at 37 ° for a complete Acm removal. Then the solution mixture was treated with 40 equiv. Cys-PEGA resin for 1 h at 37 ° and 1 equiv. of DTT. Finally, 2 equiv. of iodoacetamide at 37 ° were added to give the tri-alkylated peptide 3.2 mg of peptide 3 (1.35 × 10−4 mmol) and CSP-1-2  were dissolved in 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH ~7.2  containing 20 equiv. of MPAA and 10 equiv. of TCEP. The reaction was incubated at 37 °C for 2 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% buffer B (Acetonitrile mixed with 0.1% TFA) over 30 min. After completion of the ligation reaction, the product was treated with 100 eq. of MgCl2 dissolved in 100 µL of 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH ~7.2 at 37 °C for 10 min. For Thz removal, [Pd(AllylCl)]2  and GSH  were dissolved in 100 μl of 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH ~7.2, and added to the reaction mixture, which was incubated at 37 °C for 1.5 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% B over 30 min. After completion of the reaction, 60 equiv. of DTT was added to quench and precipitate the palladium from the reaction mixture. After centrifugation, the supernatant solution was collected and purified using semi-preparative HPLC, C4 column and gradient of 20–60% B to give the desired product in ~45% yield  and CSP-1-3  were dissolved in 100 μl of 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH ~7.2, containing 20 equiv. MPAA and 10 equiv. of TCEP at pH ~7.2 and incubated at 37 °C for 6 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% B over 30 min. After completion of the ligation reaction, CSP1-5 was isolated using semi-preparative HPLC, C4 column and a gradient of 20–60% B to give the desired product in ~50% yield,  in 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH~7.2  and incubated at 37 °C for 2 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column with a gradient of 0–60% B over 60 min. After completion of the reaction, 40 equiv. of DTT was added to quench and precipitate the free palladium from the reaction mixture. After centrifugation of the reaction, the supernatant solution was collected purified using semi-preparative HPLC, C4 column and gradient of 20–60% B to give the desired product in ~50% yield,  and H2A-2, , were dissolved in 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH~7.2  containing 20 equiv. of MPAA and 10 equiv. of TCEP. The reaction was incubated at 37 °C for 4 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% B over 30 min. After completion of the ligation reaction, the product was treated with 100 equiv. of MgCl2, dissolved in 100 µL of 6 M Gn·HCl, 200 mM Na2HPO4, pH ~7.2 at 37 °C for 10 min. For Thz removal [Pd(AllylCl)]2  was dissolved in 100 μl of 6 M Gn·HCl, 200 mM Na2HPO4, pH~7.2. and the reaction mixture was incubated at 37 °C for 1.5 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% B over 30 min. After completion of the reaction H2A-3 fragment (5.7 mg 1.1 × 10−3 mmol), dissolved in 100 μl of 6 M Gn·HCl, 200 mM Na2HPO4, pH~7.2 containing 100 equiv. of MPAA and 50 equiv of TCEP was added to the reaction and incubated at 37 °C for 8 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column with a gradient of 0–60% B over 30 min. After completion of the final ligation, 60 equiv. of DTT was added to quench and precipitate the palladium from the reaction mixture. After centrifugation of the reaction, the supernatant solution was collected and dialyzed 500 ml of 6 M Gn·HCl, 200 mM Na2HPO4 buffer at pH~7.2, using Slide-A-Lyzer dialysis cassettes for 16 h. After the dialysis, radical desulfurization reaction was performed by adding 70 mg of TCEP (0.25 M), 32 mg of VA-044 (50 equiv. per sulfur) and 100 μl of t-butyl thiol  to the reaction mixture and incubated in 42 °C for 4 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column with a gradient of 0–60% B over 30 min. After centrifugation, the supernatant solution was collected and isolated via semi-preparative HPLC, C4 column and a gradient of 20–60%B was used to isolate the product in ~17% yield,  was dissolved in 100 µL H2O and 10 µL of this solution was added to the pre-dissolved H2A-5  in 50 mM Tris buffer, pH 7.5  and incubated at 37 °C for 2 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0–60% B over 30 min. After completion of t-butyl removal, Ub(1–75)-thioester, , was dissolved in 100 µL of 6 M Gn·HCl, 200 mM Na2HPO4 buffer containing 100 equiv. of MPAA and 50 equiv. of TCEP, pH ~7.2. The reaction was incubated at 37 °C for 4 h. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0-60% B over 30 min. After completion of the final ligation reaction, 40 equiv. of DTT was added to quench and precipitate the palladium from the reaction mixture. After centrifugation, the supernatant solution was collected and isolated via semi-preparative HPLC, C4 column and a gradient of 20–60% B was used to isolate the product in ~45% yield,  dissolved in 6 M Gn·HCl, 200 mM Na2HPO4 buffer, pH ~8  and the reaction mixture was incubated 1 h at room temperature, followed by 3 h at 37 °C. Progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0-60% buffer B over 30 min. After completion of elimination reaction, the product (H2AK119DHAUb) was purified using semi-preparative HPLC, C4 column and a gradient of 20-60% B was used to give the product in ~55% yield, . The octamers bearing the H2AK119DHAUb were then reconstituted into nucleosomes with salt gradient dialysis and purified using a DEAE-5PW HPLC column (TOSOH). After purification, nucleosomes were dialyzed into NCP storage buffer  and stored at 4 °C.Histone octamers were reconstituted and purified as described previouslyDHA) probe was accomplished by reacting each component at concentrations of 6 µM and 3 µM, respectively. The reaction was done in the absence of reducing agent to avoid modification of the DHA group at 25 °C for 1 h in NCP storage buffer. Samples were then analyzed by EMSA (6% TBE gel stained with SYBR Gold), SDS-PAGE (NuPage 4–12% Bis-Tris gel stained with Sypro Ruby), and western blot (antiUb-p4d1 primary (Santa Cruz Biotech Cat # B1512) and anti-mouse IgG HRP-linked secondary  were diluted at 1:5000,  and anti-mouse IgG HRP-linked secondary  were diluted at 1:5000.The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information Files, and from the corresponding author upon reasonable request.Supplementary Information


Arabidopsis thaliana (Arabidopsis), the transcription factor PHR1 acts as a Pi central regulator. PHL1 is a homolog of PHR1 and also plays a role in maintaining Pi homeostasis. In rice (Oryza sativa), OsPHR1–4 are the orthologs of PHR1 and have been implicated in regulating sensing and signaling cascades governing Pi homeostasis.Phosphate (Pi) and Nitrogen (N) are essential macronutrients required for plant growth and development. In OsPHR3 was examined in regulating the homeostasis of N under different Pi regimes. Deficiencies of different variants of N exerted attenuating effects on the relative expression levels of OsPHR3 in a tissue-specific manner. For the functional characterization of OsPHR3, its Tos17 insertion homozygous mutants i.e., osphr3–1, osphr3–2, and osphr3–3 were compared with the wild-type for various morphophysiological and molecular traits during vegetative (hydroponics with different regimes of N variants) and reproductive (pot soil) growth phases. During vegetative growth phase, compared with the wild-type, OsPHR3 mutants showed significant variations in the adventitious root development, influx rates of 15N-NO3− and 15N-NH4+, concentrations of total N, NO3− and NH4+ in different tissues, and the relative expression levels of OsNRT1.1a, OsNRT2.4, OsAMT1;1, OsNia1 and OsNia2. The effects of the mutation in OsPHR3 was also explicit on the seed-set and grain yield during growth in a pot soil. Although Pi deficiency affected total N and NO3− concentration, the lateral root development and the relative expression levels of some of the NO3− and NH4+ transporter genes, its availability did not exert any notable regulatory influences on the traits governing N homeostasis.Here the role of OsPHR3 plays a pivotal role in regulating the homeostasis of N independent of Pi availability.The online version of this article (10.1186/s12870-018-1462-7) contains supplementary material, which is available to authorized users. Oryza sativa L.) is the main dietary staple for more than half of the 7.5 billion populations in the world, of which ~ 90% is consumed in Asia alone (www.irri.org/rice-today). United Nations raises world population forecast to 9.8 billion people by 2050 due to escalated population growth particularly in Africa and India (www.un.org). According to FAO, world agriculture will thus face the daunting task of using scarce natural resources more efficiently and adapting to climate change for producing ~ 70% more food for feeding additional 2.3 billion people by 2050 (www.fao.org). Since rice provides 27% and 20% of dietary energy supply and dietary protein intake, respectively in the developing world (www.fao.org), its sustainable production is increasingly becoming pivotal for global food security.Rice (3−) and ammonium (NH4+) with the former being the predominant form in most soils [Nitrogen (N) is a key component of important macromolecules such as nucleic acids, proteins and chlorophyll and constitutes ~ 1.5–2% of plant dry matter [monium NH+ with thst soils –8 exertest soils , 10.cis-regulatory specific sequences in their promoters [PHR1 (PHOSPHATE STARVATION RESPONSE 1), a homolog of PSR1 (PHOSPHATE STARVATION RESPONSE 1) in Chlamydomonas reinhardtii [Arabidopsis thaliana [AtFer1 promoter enriched with P1BS during Pi deficiency [AtFer1 encodes plastid-located ferritin, a protein nanocage which can store up to 4,500 atoms of Fe3+ in its interior that are released in a controlled fashion [PHR1-related genes in public databases led to the identification of PHR1-LIKE1 . Pi accumulation was significantly higher in PHR1-overexpressing transgenic lines compared with phl1 mutant, and it was significantly lower in the double mutant phr1phl1 compared with the latter, which suggested partial functional redundancy between PHR1 and PHL1 [PHL2 and PHL3 are the homologs of PHL1 and of these PHL2 play a pivotal role in regulating transcriptional response to Pi deficiency and is functionally redundant with PHR1 [PHR1 orthologs (OsPHR1–3) and together they formed a network for regulating sensing and signaling cascades governing Pi homeostasis [OsPHR4 mediates Pi homeostasis and plays a pivotal role in the regulation of downstream PSR genes [OsPHR3 is induced by Pi starvation, its mutation does not exert any significant influence on the Pi concentration and on the expression of downstream PSR genes [OsPHR3 plays a role in exerting a regulatory influence on the morphophysiological and molecular traits governing N homeostasis in a manner dependent or independent of Pi availability.Reverse genetics approaches have helped to identify several transcription factors (TFs) that extert regulatory influences on an array of functionally diverse genes involved in the maintenance of N and Pi homeostasis , 11. TFsromoters . The TF nhardtii  was posithaliana . PHR1 hathaliana . PHR1 acthaliana –21. In aficiency . AtFer1  fashion . In Arab fashion  and exer fashion . These sand PHL1 . In Arabeostasis , 27. Pi-SR genes . AlthougOsPHR3 is responsive to different forms of N irrespective of Pi regimes. The silencing of OsPHR3 triggered wide-spectrum effects on different traits during vegetative and reproductive growth phases. Availability of Pi did not exert any notable effects on OsPHR3-mediated regulatory influence on N homeostasis under different N variants and the lateral root development responses under different NO3− treatments.Here, in our study, we showed that http://www.ncbi.nlm.nih.gov/BLAST) was employed for searching the homolog of Arabidopsis AtPHL1 (At5g29000) in rice, which resulted in the identification of OsPHL1 on the chromosome 2. However, this gene has been reported in 2015, which named as OsPHR3 (LOC_Os02g04640) [OsPHL1 to OsPHR3. OsPHR3 is a MYB coiled-coil (MYB-CC) domain-containing TF (http://www.ebi.ac.uk/interpro/). OsPHR3's  orthologs are AtPHR1 and AtPHL1–3 in Arabidopsis [OsPHR1–4 in rice [OsPHR3 in the shoot and root of the wild-type rice seedlings grown hydroponically in a medium supplemented with different forms and concentrations of N   . Thus webidopsis , 16, 21  in rice –28. The O3− Fig. . Furthering Fig. . It was OsPHR3 mutants in the Nipponbare background  were obtained from the rice Tos17 insertion mutant database (https://tos.nias.affrc.go.jp)  and the last (osphr3–1) exon of OsPHR3  seedlings compared with the wild-type under both +N and -N conditions   was comparable with the wild-type irrespective of N regimes, their root biomass was significantly lower than the wild-type under both +N (~ 27–30%) and -N (~ 27–33%) conditions  were comparable with the wild-type under both H NO3− and L NO3− conditions  revealed significant reductions under H NO3− (~ 52–62%), L NO3− (~ 51–52%)   were grown in a pot soil up to the grain-filling stage  Fig. . Althougons Fig. . Root syons Fig. . However2%) Fig.  and L NHage Fig. -g. The gage Fig. , which wage Fig. , and graage Fig. . The resosphr3–1 and 3–2) were grown hydroponically under +N and -N condition for 7 d to determine the effects of the mutation in OsPHR3 on the concentrations of total N, NO3− and NH4+ in the shoot and root of the seedlings   and -N shoot (~ 11–13%), +N root (~ 30–38%) and -N root (~ 27–30%), NO3− in +N shoot (~ 26–30%), +N root (~ 19–23%) and -N root (~ 23–37%) and NH4+ in -N root (~ 60–64%)  were grown in a pot soil up to the maturity (grain harvest stage) to determine the effects of the mutation in OsPHR3 on the concentration of total N in different tissues at the reproductive stage  grown hydroponically under different N regimes  in +N root, 15NO3− (~ 49–50%) and 15NH4+ (~ 25–27%) in -N root, while the corresponding values remained comparable of 15NH4+ in the +N root  in the mutants compared with the wild-type  and fourth old leaf blade (OLB) at a five-leaf stage of the wild-type and mutants (osphr3–1 and osphr3–2) grown hydroponically under H NO3− and L NO3− condition for 7d  by exposing the N-starved oldest leaves to 15N-NO3− for 5 h  Fig. -c. Furthoot Fig. , c. Althype Fig. , d. In a 7d Fig. . Compare5 h Fig. . The mutOsPHR3 exerted significant influences on the concentrations of total N, NO3− and NH4+  [4+ (OsAMTs) [3− reductase genes (OsNia1 and OsNia2) play a role in converting NO3− to NH4+ in roots, which related to N metabolism [3− reductase genes are well known to be low during nitrate deficiency and high in nitrate-sufficiency [OsPHR3 on the relative expression levels of NO3−  and NH4+  transporter and NO3− reductase (OsNia1 and OsNia2) genes in the roots of the wild-type and the mutants (osphr3–1 and osphr3–2) grown hydroponically under +N and -N conditions , -N (OsAMT1.3) or both under +N and -N conditions (OsNRT2.3a and OsAMT1.2) . On the contrary, the relative expression levels of OsNRT1.1a and OsNia1 under +N and -N condition, and those of OsNRT2.4, OsAMT1.1 and OsNia2 under -N condition were significantly attenuated in the mutants compared with the wild-type. The relative expression level of OsAMT1.3 under +N condition was significantly augmented compared with the wild-type. The results suggested differential regulatory influences of OsPHR3 on the relative expression levels of NO3− and NH4+ transporter and NO3− reductase genes under different N regimes.Since the mutation in YLB Fig. , it rais(OsNRTs) –33 and N(OsAMTs) –36 have ficiency . Therefo3− in Arabidopsis [OsPHR3 response to different NO3− regimes depending on Pi availability, the relative expression levels of OsPHR3 under H/L NO3− + P and H/L NO3− -P conditions were detected  were grown hydroponically under various NO3− and NH4+ regimes for 10d  and L NO3− (~ 86–137%) compared with the wild-type  were grown hydroponically under +P/H NO3−, -P/H NO3−, +P/L NO3− and -P/L NO3− conditions  in the mutants compared with their corresponding wild-type  and -P/L NO3− (~ 25–33%)  Fig. . These rOsPHR3 in both shoot and root  were grown hydroponically under different Pi regimes for 2 weeks. Shoot and root were harvested and assayed for total N  and NH4+  transporter genes were assayed in the roots of the wild-type and the mutants (osphr3–1 and osphr3–2) grown hydroponically under +P and -P conditions for 3d , L NO3− and -N (in shoot and root) triggered attenuation in the relative expression levels of OsPHR3. OsPHR3 was significantly induced during Pi deficiency [OsPHR3 under L NH4+ was comparable with H NH4+ in the shoot  revealed negative regulatory influences of OsPHR3 on the developmental of the number of lateral root primordia (H NO3− and L NO3−)  and density of lateral roots (L NO3−)  compared with the wild-type  Fig. , c, and 3−) Fig. -f. Auxinbidopsis , 55. Theype Fig.  suggesteype Fig. . Howeveral roots , 57. Theeld Fig. . The resild-type .3−  and NH4+ (-N root) in osph3–1 and osphr3–2 mutants compared with the wild-type suggested positive regulatory influence of OsPHR3 on different forms and concentrations of N in a tissue-specific manner  compared with the wild-type , NOner Fig. -c. It waots Fig. -c. It isand NH4+ . The difelopment . Althougby roots , acquisie latter , 60. Theion Fig. , c. The ype Fig. . The resype Fig. -c. On thmes Fig.  and rediO3− Fig. . The dat3− (OsNRTs) [4+ (OsAMTs) [OsPHR3. OsNRT1.1a encodes a low-affinity NO3− transporter and plays a role in the accumulation of N [OsNRT2.4 is largely expressed in the base of the lateral root primordia, leaves, hull and in the vascular tissue of the anther and its expression is relatively much higher in the roots supplied with NO3− compared with NH4+ solution [3− regulated root growth and NO3− distribution [4+ transporter OsAMT1;1 has higher NH4+ permeability and exhibits better growth and higher yield under optimal and suboptimal NH4+ conditions [OsAMT1.3 also encodes a high-affinity NH4+ transporter, which is expressed predominantly in -N roots [OsPHR3 caused significant reductions in the relative expression levels of OsNRT1.1a (+N and -N conditions), OsNRT2.4 and OsAMT1.1 (-N condition) and but augmentation in the relative expression levels of OsAMT1.3 (+N condition)  and nitrite reductase (NiR), and the NH4+ derived from NO3− and/or directly acquired by the root is further assimilated into amino acids in the shoot [3− reductase genes (OsNia1 and OsNia2) were reduced in +N root (OsNia1) and –N root (OsNia1 and OsNia2) in the mutants  showed that there were several Pi related cis-elments on the promoter of OsNRT2.4, such as W-box. However, there was no P1BS, which is the PHR1-specific binding sequence [OsPHR3 may regulate the NRT genes in an indirect manner. The more detailed mechanism need further verification.This raised a question whether genes encoding for transporters for NO(OsNRTs) , 62, 63 (OsAMTs) –36 are tsolution . It playnditions . OsAMT1.ion Fig. . These rescribed , 64 withescribed , 64. Amo N and P . The cissequence . It sugg3− is present [3− and higher Pi concentrations promote flowering [OsPHR3 in various responses to different NO3− or N regimes. NO3− deficiency triggered attenuation in the relative expression of OsPHR3 in shoot and root under both +P and -P conditions , inhibitory  and stimulatory (OsNRT1.1a) in the wild-type, the mutants (osphr3–1 and osphr3–2) revealed a similar trend  in regulating Pi homeostasis under different N regimes [OsPHR3 on these genes under different N regimes irrespective of Pi regimes.Earlier studies have also shown the prevalence of an antagonistic cross-talk between signaling pathways of N and Pi in rice  and Arab present , and lowlowering . This leons Fig. . This prity Fig. -f. Althoend Fig. , b. Thesend Fig. . However regimes , 28. TheOsPHR3 is responsive to different forms of N irrespective of Pi regimes. The silencing of this gene triggered wide-spectrum effects on phenotypes during vegetative and reproductive growth phases. The analysis of total N, NO3− and NH4+ concentrations, influx rates, translocation and distribution ratio of 15N, and relative expression levels of N transport and metabolism related genes suggested that silencing of OsPHR3 regulated N homeostasis in tissue-specific manner. Further an insight into the likely roles of OsPHR3 in regulating the lateral root development under different NO3− regimes and N homeostasis independent on Pi availability were gained. These results from the study explain that availability of Pi did not exert any notable effects on OsPHR3-mediated regulatory influence on N homeostasis under different N variants and the lateral root development under different NO3− treatments. It provide a basis for further detailed characterization of the cross-talk between N and P.This study presented that Oryza sativa L. ssp. japonica cv. Nipponbare) was used in the present study. The mutants osphr3–1 (RTIM NE3007), osphr3–2 (RTIM NE3709) and osphr3–3 (RTIM NE3735) in Nipponbare background were obtained from the rice Tos17 insertion mutant database (https://tos.nias.affrc.go.jp). Homozygous mutants were identified by using a set of primers (P1-P5) in two-round semi-quantitative RT-PCR and lack of OsPHR3 transcripts validated their fidelity , CaCl2 (1 mM), MgSO4 (1 mM), Na2SiO3 (0.5 mM), K2SO4 (0.35 mM), KH2PO4 (0.3 mM), EDTA-Fe (20 μM), H3BO3 (20 μM), MnCl2 (9 μM), ZnSO4 (0.77 μM), (NH4)6Mo7O24 (0.39 μM) and CuSO4 (0.32 μM) with pH adjusted to 5.5. Seedlings were then transferred to nutrient solution containing different form and concentration of N: +N (2.5 mM), -N (0 mM), high NH4+ , low NH4+ , high NO3−  and low NO3− . These hydroponic media were maintained either under +P  or -P  condition. To inhibit nitrification, hydroponic medium containing different concentration of NH4+ was supplemented with 7 μM of dicyandiamide (C2H4N4). Plants were grown under controlled conditions .Wild-type rice (OsActin (accession number AB047313) was used as an internal control and qRT-PCR analysis was performed by using SYBR Premix Ex Taq™ II (TaKaRa) in StepOnePlus™ Real-Time PCR System (Applied Biosystems). Relative expression levels of genes were computed by 2C-ΔΔT method of relative quantification [Total RNA (~ 1 μg) was extracted from the plant tissue by using Trizol reagent (Invitrogen) and treated with RNase-free DNase (Thermoscientific). First-strand cDNA was synthesized using an oligo (dT) 18 primer and reverse transcribed using Superscript II™ Reverse Transcriptase (Invitrogen). fication . The gen4 (0.1 mM) for 1 min. Concentration of total N was determined by Kjeldahl method as described [3− and NH4+ by using a continuous-flow auto-analyzer .Different tissues were harvested and washed with CaSOescribed , while tosphr3–1and osphr3–2) were grown hydroponically in the IRRI nutrient solution for 2 weeks and then deprived of N for 3 d. Plants were rinsed in CaSO4 (0.1 mM) for 1 min and then transferred to the IRRI nutrient solution containing either 0.25 mM or 2.5 mM 15NO3−  and 0.25 mM or 2.5 mM 15NH4+  for 10 min and 24 h for their influx and distribution (shoot/root), respectively. In addition, to determine the redistribution of NO3− from N-starved old to the young leaf, wild-type and the mutants were grown to the five-leaf stage. NO3− concentration of the second leaf (old) and fourth leaf (young) of the wild-type and the mutants were analyzed. Then, the oldest leaf blade of each plant was wiped gently with a sponge and incubated in solution containing 5 mM Ca (15NO3)2 for 5 h. After the treatment, the youngest leaf blade (first) from the top was sampled after 24 h for determining 15N distribution. Plants were finally rinsed in CaSO4 (0.1 mM) for 1 min. Root and shoot were separated and frozen in liquid nitrogen. Tissues were ground to a fine powder, dried to a constant weight at 70 °C and ~ 10 mg dried tissue was analyzed using Isotope-ratio mass spectrometer (Thermo Fisher Scientific).Seedlings (3-d-old) of the wild-type and the mutants (www.spss.com). Duncan’s multiple range test at P < 0.05 was carried out for all the experiments to determine the significance between the control and treatments.Data were analyzed by ANOVA using SPSS 20 program Additional file 2:OsPHR3 mutants. (PDF 155 kb)Isolation and validation of Additional file 3:OsPHR3 affects adventitious root length. (PDF 121 kb)Mutation in Additional file 4:OsPHR3 differentially affects total N concentration in different tissues. (PDF 129 kb)Mutation in Additional file 5:OsPHR3 does not affect the lateral root development under different NH4+ regimes. (PDF 154 kb)Mutation in Additional file 6:OsPHR3 has no effect on Pi and total P concentrations under different N regimes. (PDF 161 kb)Mutation in Additional file 7:osphr3 mutant identification. (DOCX 12 kb)Primers used for Additional file 8:Gene-specific primers used for qRT-PCR. (DOCX 15 kb)


The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2–C16 and C3–C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2–C8 and C3–C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3β2-nAChR subtypes.KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3β2-nicotinic acetylcholine receptors (rα3β2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3β2-nAChRs by KTM (IC Conus. With sequence hypervariability and disulfide bond constrained scaffolds, conotoxins have a complex classification system of 28 superfamilies, segregated in part by activity on ion channels present in excitable tissues such as nerve and muscle [Conotoxins are 10–50 amino acid peptide toxins present in the venom of predatory marine snails of the genus d muscle ,4,5,6,7.α-CTxs possess rigid scaffolds and are among the most potent inhibitors of nAChRs, making them insightful molecular probes for the elucidation nAChR binding paradigms. α-CTx structure–activity relationships have been developed for α-CTxs and their analogs, leading to the discovery of disease-relevant nAChR subtypes and the identification of new ligand–nAChR binding sites ,9,10. MoDockoMatic is an open source program with an intuitive user interface to run software applications for ligand and receptor structure file creation, perform high throughput virtual screening, and output docking results ranked in order from best to worst binding affinity ,32,33,34The sequences of α-CTx MII and KTM both contain 16 amino acids B. α-CTx KTM was qualitatively evaluated for nAChR bioactivity using a PC12 cell assay; antagonist activity by KTM in the presence of acetylcholine (ACh) was confirmed , prompti50 for KTM on rα3β2 nAChRs expressed in Xenopus laevis oocytes. 50 of 0.19 ± 0.02 nM commensurate with α-CTx MII with an IC50 of 0.35 ± 0.08 nM. α-CTx MII and KTM have Hill coefficients of 0.5 and 0.7, respectively.Two-electrode voltage clamp experiments were used to determine the ICAnalysis of the circular dichroism (CD) spectrum for KTM  and alkylation  . Sequence analysis showed the disulfide bridging pattern was not consistent with the expected α-CTx C2–C8/C3–C16 globular linkage as found in α-CTx MII, but rather a C2–C16/C3–C8 ribbon linkage , as obse1H resonances for KTM was achieved using standard methods [2O were used to reduce ambiguities in assignment. Fifteen amino acid spin systems were assigned in the fingerprint region (7.6–8.8 ppm), and the final amino acid, P6, was identified in the α-proton region (5.2–3.8 ppm). NMR structure determination for KTM was performed to compare the computationally predicted C2–C8/C3–C16 globular structure to the synthesized C2–C16/C3–C8 ribbon structure. Assignment of  methods . A combiXenopus oocytes expressing rα3β2-nAChRs. KTM caused blockage of rα3β2-nAChRs with an IC50 of 0.19 ± 0.02 nM as compared to 0.35 ± 0.08 nM for α-CTx MII , resulting in many proton chemical shifts in similar or identical electronic environments. The interpretation of NMR spectra for KTM was severely complicated due to peak overlap, ring-flipping of aromatic side chains, and spectral shift between HPerhaps the most striking finding of this study was that KTM, which was predicted to act on rα3β2-nAChRs, did indeed do so, and with very high potency, despite having a ribbon-type α-CTx disulfide connectivity. There is precedent for ribbon isomers of Framework I α-CTxs with binding affinity for rα3β2-nAChRs. Dutton, et al. (2002) characterized a non-native ribbon disulfide bond isomer of recombinant α-CTx AuIB that, while more flexible than the native globular isomer, exhibited 10 times more potent activity with nAChRs in rat parasympathetic neurons than the native globular isomer ,37. They2O. The column was a Phenomenex Luna C18 with specifications of 5 µM, 100 Å, 4.6 ×250 mm. The flow rate was 1 mL/min and the injection volume was 20 µL. Peptide identity was confirmed by mass spectrometry where the expected molecular weight was 1942.21 Da and the found molecular weight was 1941.52 Da.KTM was synthesized by and purchased from CS Bio  in folded form as a white powder. Following solid phase peptide synthesis, disulfide linkages were allowed to form into the most thermodynamically stable conformation under reducing conditions. The major isomer was collected by HPLC at ca. 98.36% purity. The peptide was collected at 10.71 min using a 20%–50% gradient of Buffer B (0.1% TFA in ACN) over a 20 min run time, where Buffer A was 0.1% TFA in H18 column  (Thermo Fisher), and mobile phases consisting of 0.1% formic acid (v/v) in water (Buffer A) and 0.1% formic acid (v/v) in acetonitrile (Buffer B) with a flow rate of 0.3 mL/min. A linear gradient method beginning at 85% Buffer A and 15% Buffer B, up to 60% Buffer B over a 25 min run time achieved desired separation from a 10 µL sample injection volume. Data were analyzed with the Chromeleon 7.2 Chromatography Data System (Thermo Fisher). The partial reduction strategy of Gray [Analysis was performed using an UltiMate 3000 HPLC  equipped with a Corona Veo RS charged aerosol detector (CAD), an UltiMate 3000 Diode Array Detector (DAD), and MSQ Plus mass spectrometer (MS). HPLC separation of peptide species was achieved using an Acclaim 120 C of Gray  was usedθMRE, in deg × cm2 × dmol−1) for each spectrum was calculated from the formula θMRE = θ/(10Cr × l), where θ is the measured ellipticity in millidegrees, Cr is the molar concentration, and l is the path length in centimeters. The α-helical content was estimated from the formula θMRE = 30300fH–2340, where fH is the fraction of α-helical content  calculated from the θMRE at 222 nm, which is a widely used proxy for helical secondary structure that is useful to assess disruption of the secondary structure, as was performed here [Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter  with a cell path length of 0.1 cm at room temperature in a nitrogen atmosphere in water. Scans were acquired from 190 to 250 nm. The bandwidth of 1 nm, a speed of 50 nm/min, and a resolution of 0.5 nm were used. A total of 5 scans per sample were averaged, baselines were subtracted, and the sample was run in triplicate. A final concentration of 50 µM of each sample was used. Analysis and data processing were carried out with the Jasco system software and Microsoft Excel. Mean residue ellipticity . d-ACN was used due to the limited solubility of KTM in water. D2O samples were prepared by dissolving lyophilized sample in 30% d-ACN/70% D2O solvent and immediately acquiring spectra. Two-dimensional 1H NMR experiments and spectral interpretation were performed by established methods [NMR samples were prepared at a concentration of approximately 3 mM in either 30% deuterated acetonitrile (d-ACN)/70% water or 30% d-ACN/70% deuterium oxide . Parameter set details are provided in All NMR data were acquired on a 600 MHz Bruker Avance III NMR spectrometer at 298 K. COSY, TOCSY, and NOESY spectra were acquired using water suppression for aqueous samples. A series of NOESY spectra were acquired with mixing times of 150, 200, 250, 300, and 350 ms. TOCSY spectra were acquired with an 80 ms mixing time . The autoassign script in CYANA was used for assignment of 69 hydrogen atom chemical shift values. A chemical shift tolerance of 0.075 was used for both axes. Disulfide bonds were used as distance restraints. CYANA produced an ensemble of 20 top structures based on 32 restraints. The final set of 20 structures gave a root-mean-square deviation among backbone atoms of 1.7 ± 0.4 Å. The structure most representative of the mean structure was determined with WHATIF . Prochec® CRL1721™ PC12 cells were provided by the Biomolecular Research Center at Boise State University.Cell culture and bioactivity assessment were performed according to Marquart, et al. . The PC1PC12 cells were cultured into laminin-coated flasks and triturated to detach. Cells were grown in a T-75 flask until dense enough to plate onto a laminin-coated flat-bottom well plate and treated with starvation media  for 3–4 days before performing the assay. Cells were washed with Locke’s solution before introducing the assay solution, which included 10 µM toxin, 0.8 ng/mL POD, 25 ng/mL MAO, and 50 µM luminol. The luminol was added to quench any dissolved oxygen in the cell environment and the assay solution. Cells were allowed to equilibrate for 5–10 min before the addition of 2 mM luminol and 50 µM ACh, for a total well volume of 100 µL. Upon equilibration, the luminescence signal was detected. (-)MAO and (-)cells were used as negative controls. Assays were performed at 37 °C, detecting luminescence for 1–2 min per well. Luminescence response curves were then integrated as a measure of dopamine secretion inhibition resultant from peptide treatment for analysis.Xenopus according to protocols approved by the Institutional Animal Use and Care Committee at ISU and according to AAALAC guidelines. Oocytes were isolated with 2 mg/L collagenase and cultured at 17.5 °C in sterile ND-96 saline containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 1 mM MgCl2, and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with 5 mM Na pyruvate, 2% horse serum, 100 mg/L gentamicin, 100 mg/L amikacin, 50 mg/L ciprofloxacin, 20 mg/L tetracycline, and 100 U/L streptomycin/penicillin, at pH 7.4. Messenger RNA was transcribed as detailed previously; α3 and β2 mRNA was coinjected at 41 nL into oocytes 3 to 5 days prior to recordings.cDNA preparation, oocyte harvest, expression of nAChR subunits, and culture were performed as described previously . The ratn is the Hill coefficient and IC50 is the inhibitory concentration at half-maximal block, by non-linear regression analysis using Igor Pro 6.37 .To record the response to ACh, oocytes were impaled with glass electrodes containing 3 M KCl for two-electrode voltage clamp electrophysiology. Oocytes were placed in a 300 µL recording chamber and perfused at 1.0 mL/min with ND-96 saline containing atropine sulfate (1 mM), at room temperature. Voltage clamp was performed with an OC-725C amplifier  with data acquisition achieved through an ITC-18 interface and PatchMaster 2.35 software . Oocytes were held at -70 mV between trials, and at -80 mV during trials of ACh application. During perfusion, a 20 to 50 ms pulse of ACh chloride  in the bath solution was locally applied from a glass capillary tube positioned above the oocyte, with pressure ejection using a PicoSpritzer II valve controller . Once a reliable response was obtained, the chamber was perfused with α-CTx MII or KTM for 15 min, and the ACh pulse was applied. Inward sodium current amplitudes for each toxin trial were normalized against the control response to ACh as percent response. Each data point of the dose response curve represents the average value of 8 to 12 measurements. Dose-dependent response curves were fit to Equation (1):Molecular dynamics simulations were performed with the AMBER03 force-field parameter set  using thThis investigation sought to validate the accuracy and usefulness to rationally design a selective antagonist of a specific nAChR isoform based on the well characterized marine drug α-CTx MII. Synthetic KTM peptide was confirmed to be a potent sub-nanomolar inhibitor of rα3β2 nAChRs. It was expected that KTM would fold into the same C1–C3 and C2–C4 disulfide pattern, consistent with α-CTx MII. Instead, the folding of KTM resulted in a C1–C4 and C2–C3 ribbon Framework I α-CTx, consistent with the recombinant isomer of α-CTx AuIB ,37. It h


Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.Chromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that   •ChOR-seq determines histone PTM occupancy on newly replicated DNA•Histone PTM positional information is preserved through parental histone recycling•Parental H3K27me3 domains are stable and inherited to daughter cells•Restoration of histone PTM levels follows mark- and locus-specific kinetics Histone modifications are a core component of the epigenome. Reverón-Gómez et al. develop ChOR-seq to profile histone modifications after DNA replication and find that the genomic localization of modified parental histones is preserved on daughter strands while new histone modification to restore pre-replication levels follows mark- and locus-specific kinetics. The organization of eukaryotic genomes into chromatin influences all DNA-based processes, including gene expression and DNA repair. Chromatin organization is particularly important for establishing and maintaining cell-type-specific transcriptional programs and thus underlies epigenetic cell memory . HoweverThe nucleosome is the basic unit of chromatin, in which 146 base pairs of DNA are wrapped around a histone core composed of a central histone H3-H4 tetramer flanked by two histone H2A-H2B dimers. Histones are decorated with a large variety of post-translational modifications (PTMs) that contribute to the establishment and maintenance of active and repressed chromatin states . Many ofin vitro replication of simian virus 40 (SV40) mini-chromosomes have shown that 1–2 parental nucleosomes are destabilized ahead of the replication fork  are mixed in a 1:1 ratio on newly replicated DNA  and thatde novo histone methylation after DNA replication increases the level of the marks within regions already demarcated by modified parental histones. Notably, we find that restoration of histone PTM levels follows mark- and locus-specific kinetics, arguing that the epigenome is undergoing complex changes across the cell cycle that could underlie cell-to-cell heterogeneity.To understand how the PTM landscape is duplicated during DNA replication, it is necessary to elucidate where and when modified histones are deposited within a given genomic locus post replication. We have developed a technology to analyze chromatin occupancy after DNA replication by next-generation sequencing, termed ChOR-seq. ChOR-seq can track the occupancy of proteins and histone PTMs after replication fork passage genome-wide. Given that newly synthesized histones are devoid of tri-methylation marks at the time of deposition , ChOR-se. To optimize coverage of transcriptionally active and repressed loci, we labeled replicating DNA in early S phase and mid S phase, respectively, corresponding to the replication timing of these regions (Drosophila S2 cells (ChOR-seq is based on short (10–20 min) pulse labeling of replicated DNA with a nucleotide analog (EdU) followed by sequential chromatin immunoprecipitation (ChIP) of a specific histone PTM. Labeled DNA is then biotinylated via Click-IT and isolated by biotin-streptavidin pull-down prior to analysis by next-generation sequencing A. To inv regions . We harv regions A. FinallS2 cells A  with replicated DNA profiles (EdU pull-downs) E. This w omitted G and S1HTo address how accurately histone PTM profiles are copied during replication, we compared occupancy patterns of four modifications—H3K27me3, H3K4me3, H3K36me3, and H3K79me3—in pre-replicative and nascent chromatin by parental ChIP-seq and ChOR-seq, respectively. Locally, we observed that histone modification patterns were preserved during replication A. PlottiDrosophila chromatin spike-in to normalize read counts, revealing quantitative differences in signal between samples that are lost with conventional data processing methods. We therefore carried out H3K4me3 qChOR-seq on nascent chromatin and on mature chromatin harvested 1 hr later, with cells still in S phase (T1); 6 hr later, when cells reached G2/M but had not passed through mitosis (T6); and 12 hr later, when cells had passed through mitosis and were in G1 of the next cell cycle (T12) (Drosophila chromatin (RRPM) revealed a substantial accumulation of H3K4me3 during the first 6 hr of chromatin maturation , which takes advantage of a le (T12) A. While le (T12) B, normalturation B.Figure Genome-wide, we observed marked gains in H3K4me3 signal between T0 and T1 and T1 and T6 but no further increase between T6 and T12 C and 3D.The advantage of qChOR-seq is that it provides both quantitative and positional information about PTM signal. We therefore next asked whether H3K4me3 restoration occurs with different kinetics in different parts of the genome. To address this, we compared qChOR-seq signal across our time course and considered a locus restored when it reached H3K4me3 levels close to that observed at the 12-hr time point . This reWe have previously defined the restoration kinetics of H3K27me3 at the global level using quantitative mass spectrometry . Bulk maTo identify genomic loci with differential restoration rates, we parsed all replicated H3K27me3 regions according to when the maximal H3K27me3 level was reached. We considered a region restored when reaching a level close to that observed at the 24-hr time point . With thde novo histone H3 K27 tri-methylation to the inheritance of H3K27me3 to daughter cells, we therefore performed H3K27me3 qChOR-seq analysis in the presence of an inhibitor of EZH2  and across mitosis to daughter cells . H3K27me3 nascent qChOR-seq revealed that the H3K27me3 occupancy patterns in nascent chromatin were largely unaffected by lack of EZH2 activity A. The dede novo H3K27 methylation .Further information and request for resources and reagents should be directed to and will be fulfilled by the Lead Contact Anja Groth (2. Drosophila S2-DRSC cells were obtained from the Drosophila Genomics Resource Center. S2 cells were grown in suspension in spinners in M3+BPYE (Sigma-Aldrich) media with 10% heat-inactivated FBS, penicillin and streptomycin at 25°C.Human HeLa S3 cells  were obtained from ATCC and were grown in suspension in spinners. Cells were grown in DMEM medium (Thermo Fisher Scientific) supplemented with dialyzed FBS (Invitrogen), penicillin, and streptomycin at 37°C and 5% COFor all ChOR-seq and ChIP-seq, HeLa S3 cells were synchronized at the G1/S border by a single thymidine block  and released into fresh media containing deoxycytidine (24 μM) for the indicated amount of time. For H3K4me3, H3K36me3 and H3K79me3 analysis, parental samples were collected immediately after release. 2 hr and 15 min later (early S), DNA was labeled for 10 min with medium containing 5-ethynyl-2′-deoxyuridine . Immediately following the EdU label, nascent chromatin was collected. To study H3K4me3 maturation, following EdU labeling and collection of a subset of cells as the “nascent” sample, remaining cells in the spinner culture were washed with PBS, resuspended in new medium containing deoxycytidine (24 μM), and incubated at 37°C until collection at the indicated time point.For H3K27me3 analysis, the same procedure as above was followed, but with different time points. The parental sample was collected 2 hr and 15 min after release from the thymidine block. 1 hr later (mid S), DNA was labeled for 20 min with medium containing EdU (10 μM). Immediately following the EdU label, nascent chromatin was collected. To study H3K27me3 maturation, following EdU labeling and collection of a subset of cells as the “nascent” sample, remaining cells in the spinner culture were washed with PBS, resuspended in new medium containing deoxycytidine (24 μM), and incubated at 37°C until collection at the indicated time point. To prevent cells from entering into a second round of replication, thymidine (2mM) was added to the medium 10 hr post-EdU label and cells were incubated until reaching 24 hr post-EdU label. For experiments using EZH2 inhibitor  , inhibitDrosophila S2 chromatin was labeled with EdU (10 μM) for 39 hr for use as an internal control. For Drosophila chromatin and newly synthesized DNA was labeled with biotin-dUTP as described in For qChOR-seq experiments, asynchronous Drosophila S2 cells were fixed, lysed, and sonicated as described above. After sonication, HeLa S3 input chromatin was mixed with Drosophila S2 chromatin .In ChOR-seq, ChIP-seq and replicated DNA isolation, HeLa S3 cells were immediately fixed in 1% formaldehyde. Then, glycine was added to a final concentration of 0.125 M and the reaction was incubated for 5 min at room temperature. Fixed cells were lysed for 20 min in ice-cold lysis buffer  supplemented with leupeptin, aprotinin, pepstatin, and PMSF. Lysates were passed through a 21G needle and sonicated using a Bioruptor nextGen (Diagenode) with the following settings: 20 cycles, 30 s ON, 30 s OFF, high intensity. Sonicated chromatin was centrifuged at 14,000 rpm at 4°C for 10 min and the supernatant was isolated for subsequent steps. In parallel, Drosophila S2 sonicated chromatin were diluted up to 500 μL with dialysis buffer  and 400 μL of incubation buffer  supplemented with leupeptin, aprotinin, pepstatin, and PMSF. Chromatin was pre-cleared with Protein A agarose beads for 1 hr at 4°C. After pre-clearing, chromatin was incubated with the corresponding antibody  overnight at 4°C, followed by incubation for 3 hr with pre-blocked Protein A agarose beads (incubated in 1 mg/ml BSA in RIPA buffer overnight). Chromatin bound to beads was washed three times in RIPA buffer , once in RIPA buffer with 0.5 M NaCl, once in LiCl buffer  and twice in TE . Chromatin was incubated with RNase A for 30 min at 37°C. SDS was then added to a final concentration of 0.5% and samples were incubated with proteinase K (1 mg/ml) for 10 hr at 37°C followed by 6 hr incubation at 65°C for de-crosslinking. DNA was purified using the MinElute PCR purification kit (QIAGEN). Finally, 10 ng of purified DNA was subjected to end repair, A-tailing and amplification using the KAPA Hyperprep kit protocol (Roche). Before amplification, DNA was size-selected with Agencourt AMPure XP beads (Beckman Coulter) to obtain fragments between 200-700 bp. For amplification, 6 PCR cycles were used followed by clean-up with Agencourt AMPure XP beads.Parental ChIPs were performed as described in Drosophila S2 mixed chromatin was subjected to standard ChIP as described above. 100 ng of immunoprecipitated DNA or de-crosslinked input material from labeled chromatin was then subjected to end repair, A-tailing, and adaptor ligation using the KAPA Hyperprep kit following manufacturer’s instructions, except that 1.25 μM of Illumina-compatible indexed adapters (Pentabase) were ligated to A-tailed DNA for 60 min at 20°C and cleaned-up with Agencourt AMPure XP beads. For qChOR-seq experiments, indexed DNA from all time points in the same time course were then mixed together. Then, Click-IT was performed on 200 ng of indexed and mixed DNA for 30 min at room temperature under the following conditions , 5 mM aminoguanidine (Sigma-Aldrich), and 10 mM sodium ascorbate. DNA fragments between 200-700 bp were size-selected using Agencourt AMPure XP beads and resuspended in TE. Next, to capture biotinylated products, MyOne Streptavidin T1 beads (Thermo Fisher Scientific) were washed three times with 1X B&W buffer  Tween-20) and resuspended in 2X B&W buffer at a volume equal to the volume of biotinylated DNA. Streptavidin beads were then mixed with biotinylated DNA and rotated for 30 min at room temperature. Beads containing biotinylated DNA were washed four times with 1X B&W buffer, twice with 1X TE with 0.05% (V/V) Tween 20, and once with 10 mM Tris-HCl pH 7.5. Finally, beads were resuspended in double distilled water. PCR amplification of ChOR-seq samples was performed following the KAPA Hyperprep kit protocol using the streptavidin bead suspension as a template (10-15 cycles of PCR). Following PCR, streptavidin beads were purified using a magnetic rack, and the supernatant was cleaned-up with Agencourt AMPure XP beads.For ChOR-seq experiments, 30 ug total of sonicated HeLa S3 and Drosophila chromatin, 120-240 μg of HeLa S3 chromatin was used per condition and streptavidin pull-downs were done on chromatin instead of on purified DNA. After the last wash with TE during ChIP, chromatin was separated from Protein A agarose beads by an incubation with elution buffer  for 30 min at 37°C in rotation. The supernatant was 1:20 with RIPA buffer and chromatin was then incubated with MyOne Streptavidin T1 beads to perform streptavidin pull-downs as described in In ChOR-seq experiments with biotin-dUTP performed without exogenous https://seqcenter.ku.dk) and at the Biotech Research and Innovation Centre (BRIC) (https://www.bric.ku.dk) using Illumina HighSeq 4000 and NextSeq 500 machines to obtain 50 bp and 75 bp single-end reads, respectively. Reads were aligned to the February 2009 human genome assembly (GRCh37/hg19) and the April 2006 D. melanogaster genome assembly (BDGP R5/dm3) by Bowtie (Drosophila unique mapped reads to get quantitative information in the form of reference-adjusted RPM (RRPM) as described in ChOR-seq, ChIP-seq, replicated DNA and input samples from two independent time course experiments for each histone mark were sequenced at the Danish High-throughput DNA Sequencing Centre  ratios greater than 1.5-fold and present in both replicates were considered for analysis. CpG islands overlapping R1 and R6 regions were respectively assigned to that region for analysis; CpG islands overlapping both a R1 and a R6 region were discarded. To ensure the CpG densities, calculated for coordinates in the hg18 genome assembly, remained accurate, R1 and R6 region coordinates were converted from hg19 to hg18 using UCSC LiftOver before defining overlaps.To study the restoration dynamics of H3K27me3, reads were summed in 2 kb bins that overlapped with both parental H3K27me3 peaks and replicated regions. Regions were then classified into R0, R4, R10 and R24 categories according the time they need to reach T24 H3K27me3 levels. Only bins with R24/R(X) ratios greater than 1.5-fold and present in both replicates were considered for analysis.For analysis of the H3K27me3 loss rate in the presence of EZH2 inhibitor, H3K27me3 loss categories were defined as the 2 kb windows present in replicated H3K27me3 parental peaks that showed a T0/T24 fold change smaller than 1.5 (low), between 1.5 and 3 (moderate) and bigger than 3 (high). Only bins that fit the criteria in the two independent replicates were considered for the analysis.For analysis of cell cycle progression, synchronized cells were fixed with 70% ethanol and labeled with propidium iodide (10 μg/mL) for 30 min in the dark, before analysis on a FACSCalibur machine. FACS profiles were analyzed by FlowJo 10.0.8 software.The statistical tests applied in this study are stated in the figure legends and were calculated using custom R scripts. In GSM923449 (GSM733696), H3K4me3 (GEO: GSM733682), and EZH2 (GEO: GSM1003520) were taken from GSM788633) was obtained from GSM958735) (GSE110354.Replication timing was obtained from Repli-seq datasets GEO: SM923449 . ChIP-seM958735) . Positio


Ferula  were evaluated.Histone deacetylase inhibitory and cytotoxic activities of 18 naturally occuring terpenoids , coumarins  and sulfur-containing derivatives  from the roots of three species of The cytotoxic activity of compounds was evaluated against human cancer cell lines  by AlamarBlue® assay using vorinostat as the positive control. On the other hand, we aimed to evaluate their inhibitory activities against pan-HDAC.F. flabelliloba was subjected to silica gel column chromatography. Further purification by preparative thin-layer chromatography (PTLC) and semipreparative RP-HPLC yielded twelve known compounds (1-12). This is the first report on the isolation of guaiol (1), persicasulphide C (3) and conferoside (10) from the roots of F. flabelliloba. Six compounds including persicasulfide A, conferone, feselol, latisufide C, conferoside and ferutinin showed cytotoxic activity with IC50 values in the range of 11.61-49.40 μM against cancer cells and pan-HDAC inhibitory activity with IC50 values in the range of 1.06-35.27 μM.The methanolic extract of the roots of 50 values in the range of 1.06-10.73 μM. Conferone was more active than others with a higher potency for HDAC inhibition (1.06- 1.17 μM).Results indicated that persicasulfide A (2), conferone (6) and feselol (7) showed moderate cytotoxicity with IC50 values in the range of 11.76-39.24 μM against cancer cells and potent pan-HDAC inhibitory activity with IC Acetylation process is an important post-translational modification that regulated by the activity of two families of enzymes with antagonistic functions: histone acetyl transferases (HATs) and histone deacetylases (HDACs) . Recent Ferula is a large genus of perennial herbs belongs to the Apiaceae family which includes some 180 species  spectra were obtained using Bruker AVANCE-300 spectrometer . Chemical shifts are given as δ (ppm) using TMS as an internal standard. Analytical TLC was performed on silica gel 60 F254 . Preparative TLC was performed on silica gel 60 GF254 . Column chromatography (CC) was conducted with Si gel 230–400 mesh . Observation of plates was carried out under UV CAMAG spectrometer  at 254 nm. Semipreparative HPLC was performed on a KNAUER liquid chromatograph system with a quaternary pump (Smartline Pump 1000) and semi-prep c18 column . Diode array detector (Smartline DAD 2800) and EZ Chrom Elite software was used for detection and processing of data, respectively.Plant materialF. flabelliloba were collected and identified by Dr M Iranshahi, in May 2014, from Khorasan-Razavi province, Golmakan. A voucher specimen (No. 13064) was deposited in the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences.The roots of Extraction and isolationF. flabelliloba (250 g) were powdered and extracted three times with MeOH  using maceration method at ambient temperature. TheMeOH extracts were combined and the solvents evaporated under vacuum pressure leaving 50 g of a brown residue. Part of the extract (35 g) was subjected to silica gel chromatography (55×5 cm) using PET: EtOAc and EtOAc- MeOH [ × 2 L] as a gradient solvent system. Thirty eight fractions were collected and combined into eight major fractions on the basis of their TLC profiles: A , B , C , D , E , F , G , and H . Fractions A–D were further purified using PTLC. Finally, compound 1  and 2  was obtained from fraction A using DCM system. Compound 3  was obtained from fraction C using PET: EtOAc (4:1.5) system. Fraction B afforded off-white crystals of compound 4 (130 mg). Compound 5  was obtained from fraction D using DCM: MeOH (10:1) system. Fraction E afforded white crystals of compound 6 (148 mg). Fraction F afforded white crystals as an inseparable mixture of Compounds 11 and 12 (77 mg). Fraction G was separated and further purified by Semipreparative HPLC with a gradient of MeOH–H2O on a semi-prep C18  column to yield compounds 7 , 8  and 9 . Fraction H was subjected to silica gel CC (5 × 80 cm) by gradient polarity of  DCM: MeOH  to afford compound 10 . The structures of the compounds were assigned via comparison of 1H- and 13C- NMR spectra with those of previous reports . Latisufides (A-E) (13-17) were isolated and described in our previous report (18-20) were isolated from the roots of F. ovina as previously described , ovarian (A2780), and lung (A549) cancer cells, obtained from Biotechnology Laboratory stocks, Biotechnology Research Center  and colon (HCT116) obtained from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran, Tehran (Iran). All cells were grown in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% fetal bovine serum , penicillin (100 units/ml) and streptomycin (100 mg/ml) in an atmosphere of 95% OCytotoxicity assayIn vitro cytotoxicity tests were performed using a nonfluorescent substrate, AlamarBlue®  cell viability assay as described with some modifications (16). Four cell lines  were grown in RPMI-1640 medium at 37 °C in a 5% CO2 incubator. After counting, the cells were seeded in 96-well microplates (1 × 104 cells/100 µl in each well) and cultivated for 24 hr before addition of test compounds. Triplicate plates for three wells of each concentration were seeded. The test compounds, were dissolved in dimethyl sulfoxide . Then, the cells were treated with the different concetrations of the test compounds , positive control, negative control and cell culture medium as blank for 48 hr in a 5% CO2 incubator. Thereafter, 10 μl of AlamarBlue® reagent was added and incubated for another 4 hr. Then, absorbance was measured at 600 nm using an ELISA microplate reader . IC50 values were calculated by nonlinear regression analysis using GraphPad prism (Version 6.0) software. Positive control was vorinostat .Whole-cell HDAC inhibition assayet al. (4 cells/90 µl culture medium per well). Three wells defined as 100% initial activity and three wells defined as background and three wells for each concentration were seeded. After 24 hr, the cells were further incubated for 18 hr with increasing concentrations of test compounds, positive control vorinostat and cell culture medium alone as negative control at 37 °C with 5% CO2. The test compounds, were dissolved in dimethyl sulfoxide  and their concentrations adjusted to 70, 40, 20, 5, 1 μM through dilution with the growth medium. The first step of reaction was started by adding 10 µl of 3 mM Boc-Lys (ε-Ac)-AMC  to each well to reach a final concentration of 0.3 mM. Then, cells were incubated with the Boc-Lys (ε-Ac)-AMC for 3 hr under cell culture conditions. After this, 100 µl/well of stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% NP40, 2.0 mg/ml trypsin, 10 µM vorinostat) was added and the mixture was developed for another 3 hr under cell culture conditions. The final volume of the solution (210 µl per well) were transferred to the black 96-well microplate with flat clear bottom. We calculated fluorescence intensity at an excitation of 360 nm and emission of 470 nm in a NOVO star microplate reader .The HDAC assay was based on an assay by Marek et al.  with minTrypsin inhibitory assayet al. (2) and added (except for blanks) to each microplate well. Test compounds were dissolved in DMSO and added at a final concentration of 70 μM (1% DMSO) in each well. The reaction initiated when BOC-Lys-AMC substrate  was added. Then, plate was incubated for 45 min. The amount of trypsin inhibition was calculated by comparing the amount of deacetylated substrate between control and test compounds. We calculated the relative amounts of deacetylated substrate by fluorescence reading with excitation at 360 nm and emission at 470 nm. (Caffeic acid was as positive control).Trypsin inhibition assay was carried out based on the method of Zwick et al.  with somF. flabelliloba were subjected to silica gel column chromatography. Further purification for collected fractions developed by the preparative thin-layer chromatography (PTLC) and semipreparative RP-HPLC yielded twelve known compounds (1-12). A sesquiterpenoid alcohol, guaiol (1), two sulfur-containing compounds, persicasulphide A (2) and C (3), the prenylated coumarin umbelliprenin (4), five sesquiterpene coumarins, farnesiferone B (5), conferone (6), feselol (7), ligupersin A (8) and conferdione (9) and inseparable mixture of farnesiferol B and lehmferin  together with a sesquiterpene coumarin glycoside, conferoside (10).The MeOH extract from the roots of 1-12 , conferone (6), feselol (7), latisulfide C (15), conferoside (10) and ferutinin (18) possessed moderate cytotoxicity against the cancer cell lines with IC50 values in the range of 11.61-49.40 μM. These compounds showed pan-HDAC inhibitory activity with IC50 values in the range of 1.06-35.27 μM. The most potent cytotoxic test compound was persicasulphide A (2) with IC50 values of 11.61, 11.76, 15.35 and 19.38 μM against HCT116, HeLa, A549 and A2780 respectively. The test compounds were subjected to an in vitro trypsin inhibition assay to test for interference with the assay by direct trypsin inhibition. According to this assay, 13 compounds were evaluated at a maximum concentration of 70 μM and did not display any inhibition toward trypsin . These results suggested that the HDAC inhibition measured was not due to the protease inhibition and the fluorescence-based assay clearly reflect the real HDAC activity.Our findings revealed that six tested compounds persicasulfide A (2), latisulfide C (15) and latisulfide E (17) probably responsible for their biological activities via disulfide exchange mechanism. Among sulfur-containing derivatives, persicasulphide A from F. flabelliloba showed the most HDAC inhibition and cytotoxic activities. Disulfides as thiol oxidizing agents, able to  oxidize thiolate cysteine residues on proteins to form mixed disulfides, thereby affecting protein or enzyme function and leading to cell death  may contribute to the cancer chemoprotective properties  , conferone (6), feselol (7), ligupersin A (8), conferdione (9) and conferoside (10) were identical; differences in cellular effects were most likely due to the presence of hydrogen bond, arene-arene and hydrophobic interactions that may be specific to active site of the histone deacetylase enzyme. Previous investigations revealed a cytotoxic effect of conferone on human colorectal adenocarcinoma, ovarian carcinoma, lung cancer, melanoma and renal cell lines    was the ll lines -28. In tin (Pgp) . In addiin (Pgp) -31 have Peptidase inhibitory activity of test compounds may be considered as one of the main explanation for the relatively potent HDAC inhibition of compounds in contrast to their less pronounced cytotoxicity. Although, results could be affected by this effect and numerous natural products are already known to inhibit trypsin , 33, our2), conferone (6) and feselol (7) showed moderate cytotoxicity with IC50 values in the range of 11.76-39.24 μM against cancer cells and potent pan-HDAC inhibitory activity with IC50 values in the range of 1.06-10.73 μM. Conferone was more active than others with a higher potency for HDAC inhibition (1.06- 1.17 μM).In the present study persicasulfide A (Supporting InformationNMR spectra and spectral data for all compounds tested in this study are shown in Figure S1- 16 and Table S1. Column graphs of dose-dependent growth inhibition are shown in Figure S17. Trypsin inhibition of test compounds in the trypsin inhibitory assay are shown in Figure S18.The authors declare that there are no conflicts of interest.


Although the levels of vitamin D2 in UV-exposed mushrooms may decrease with storage and cooking, if they are consumed before the ‘best-before’ date, vitamin D2 level is likely to remain above 10 μg/100 g fresh weight, which is higher than the level in most vitamin D-containing foods and similar to the daily requirement of vitamin D recommended internationally. Worldwide mushroom consumption has increased markedly in the past four decades, and mushrooms have the potential to be the only non-animal, unfortified food source of vitamin D that can provide a substantial amount of vitamin D2 in a single serve. This review examines the current information on the role of UV radiation in enhancing the concentration of vitamin D2 in mushrooms, the effects of storage and cooking on vitamin D2 content, and the bioavailability of vitamin D2 from mushrooms.When commonly consumed mushroom species are exposed to a source of ultraviolet (UV) radiation, such as sunlight or a UV lamp, they can generate nutritionally relevant amounts of vitamin D. The most common form of vitamin D in mushrooms is D Vitamin D stimulates the synthesis of the calcium transport proteins in the small intestine, enhancing the absorption of dietary calcium and thereby reducing the risk of osteomalacia in adults and rickets in children ,2. Adequ2, found in fungi and yeast, and D3, found in animals; lesser amounts of vitamin D3 and D4 are also found in fungi [2) [The two main dietary forms of vitamin D are Din fungi ,10,11,12in fungi ,14. Sun-ungi [2) ,16,17. V2 content and to examine whether the amount of vitamin D2 generated was nutritionally significant. We focussed on the three most commonly consumed mushrooms worldwide: the button mushroom Agaricus bisporus (Lange) Imbach (30% of worldwide consumption), oyster mushrooms Pleurotus (Jacquin) Kummer , and shiitake mushrooms Lentinula edodes (Berkeley) Pegler (17% of worldwide consumption), together comprising approximately three-quarters of all mushrooms consumed [This review addresses the potential of mushrooms as a good dietary source of vitamin D. We considered mushrooms exposed to different sources of UV radiation  to gauge the potential for increasing vitamin Dconsumed . StudiesThe recommended intake of vitamin D is 5–15 μg/day 200–600 IU) in Australia and New Zealand, depending on age , 15–20 μ0–600 IU 3 and its hydroxylated metabolite 25-hydroxyvitamin D3 (25(OH)D3) [Estimates of the dietary intake of vitamin D in the USA are 5–6 μg/day in adult males and 3.5–4.5 μg/day in adult females, although the intake of those taking vitamin D supplements may reach the Adequate Intake (AI) . Canadia5(OH)D3) . The discrepancy between actual and recommended vitamin D intakes indicates that dietary sources alone are unlikely to lead to adequate vitamin D status.2 in mushrooms was first reported in the early 1930s [There are five biological kingdoms: Animalia, Plantae, Fungi, Protista , and Monera  . Mushrooly 1930s .2, which is then thermally isomerised in a temperature-dependent process to ergocalciferol, commonly known as vitamin D2 [4  from mushrooms is converted to vitamin D4 [4, making them a potential source of vitamin D4 if exposed to UV radiation [2 and D4 content in UV-irradiated mushrooms [When exposed to UV radiation, ergosterol in the mushroom cell wall is transformed to pre-vitamin Dtamin D2 ,32. Throtamin D4 . All comadiation . In geneushrooms .2 content of mushrooms began with the discovery that wild edible Finnish mushrooms, the funnel chanterelle (Cantharellus tubaeformis (Bulliard) Fries), sampled in late summer and early autumn provided 3–30 μg D2/100 g fresh weight (FW), compared with less than 1 μg D2/100 g FW in the button mushroom purchased from retail outlets [2 have been found in wild funnel chanterelles (21.1 μg D2/100 g FW), Cantharellus cibarius (Fries) (10.7 μg D2/100 g FW), and Boletus edulis (58.7 μg D2/100 g FW) [2 (1.5 μg/100 g FW) was reported in wild Agaricus species in Denmark [The recent interest in the vitamin D outlets . Since t00 g FW) . A small Denmark .2 content of retail fresh button mushrooms sold around the world is commonly reported to be less than 1 μg/100 g FW [2. The National Nutrient Database of the United States Department of Agriculture lists shiitake, white button, and oyster mushrooms as all containing less than 1 μg/100 g FW of vitamin D2 [Most fresh retail mushrooms sold in the UK, Europe, North America, Australia, and New Zealand, especially the button mushroom, are grown in atmospherically controlled growing rooms, then harvested and taken to market and retail outlets in refrigerated transport. As they are usually grown in darkness, the only time they are likely to be exposed to light is during picking under fluorescent lights, which usually emit little or no UV radiation. Hence, the vitamin D100 g FW ,34,36,37tamin D2 .2, usually in excess of 10 μg/100 g FW [2 generated depends on the time of day, season, latitude, weather conditions, and exposure time. Since these mushrooms have a higher surface area to volume , sun-exposed sliced mushrooms produce more vitamin D2 than whole mushrooms from the same amount of UV radiation exposure [2 content of sliced mushrooms was as high as 17.5 μg/100 g FW after 15 min of sun exposure and reached 32.5 μg/100 g FW after 60 min of sun exposure [2 content after exposure to the midday winter sun in July in Sydney . Sun exposure to a single layer of small button mushrooms was sufficient to generate 10 μg D2/100 g FW after 1 h, while large button mushrooms took 2 h to generate the same amount of vitamin D2.When fresh button mushrooms are deliberately exposed to midday sunlight for 15–120 min, they generate significant amounts of vitamin D100 g FW ,35,39,40exposure ,39,40. A2 is to expose mushrooms to specific, controlled levels of UV radiation via a fluorescent UV lamp or a pulsed UV lamp. Mushrooms will generate vitamin D2 in response to exposure to UV radiation both during growing phase and post-harvest; however, commercial growers use UV lamps post-harvest for practical reasons. Fresh mushrooms, when deliberately exposed to a UV radiation source post-harvest, will generate significant amounts of vitamin D2 often reaching 40 μg/g dried mass (DM) (ca 320 μg/100 g FW) [2 in mushrooms is UV-B radiation (280–315 nm) [2 concentrations in all cases [An efficient way to produce nutritionally relevant amounts of vitamin D00 g FW) ,43,44,45–315 nm) ,43,46,47–315 nm) ,49, howell cases . 2 when exposed to UV-B radiation than the cap or stalk [2 compared to the cap (22.8 μg/g DM vs. 5.2 μg/g DM) [2 than shiitake at the same UV exposure level [2 concentration: 90 min of exposure to UV-B radiation at 1.14 W/m2 at 28 °C were the optimal conditions for generating vitamin D2, producing 240 μg/g DM [4 concentration in oyster mushrooms from 0 to 20 μg/g DM at 20 °C after only 30 min [In fresh shiitake mushrooms, ergosterol concentrations are highest in the gills, followed by the cap and stalk, with the gills having twice the concentration of ergosterol as the cap ,50. Subsor stalk , with thμg/g DM) . Whole ore level ,46: whenre level ,46,51,522 production has not been investigated in detail, although two studies suggest that temperatures between 25 and 35 °C may be ideal for commercial purposes. One study showed that vitamin D2 production in whole oyster mushrooms increased from 152 μg/g DM to 178 μg/g DM as the temperature increased from 15 °C to 35 °C [2 in shiitake mushrooms occurred at 35 °C and 78% moisture, producing ca. 50 μg/g DW [The influence of temperature on vitamin D μg/g DW .2 (10 μg/100 g FW) in whole mushrooms can also be effectively achieved with a commercial pulsed UV lamp within a very short time period of 1–2 s (3–6 pulses) [2 using a UV fluorescent lamp. Therefore, pulsed UV radiation may be the most efficient method of increasing vitamin D2 concentrations in mushrooms. In button mushrooms, three pulses (1 s) of UV radiation generated 11.9 μg D2/g DM [2/g DM [2 (27 μg/g DM) was reached after 12 pulses (4 s) [2 deep within the ‘flesh’ of the mushroom. The mushrooms received either 1.1 J/cm2 per pulse [2 per pulse [2 generated depends on the type and orientation of the mushrooms, whether they are sliced or whole, the distance from the lamp housing, the size of the mushroom, and the total number of pulses received. As demonstrated in two studies, nutritionally relevant concentrations of vitamin D pulses) ,42. In c [2/g DM . The maxer pulse  or 0.7912. Commercial dried mushrooms have a much longer shelf life than fresh mushrooms, often with a ‘best-before’ date of 2–3 years after packaging. They have about 15% of the original weight of fresh mushrooms, making them cheaper to transport and, potentially, a cheaper source of vitamin D2 and ergosterol content of 35 species of dried mushrooms sold in China revealed they contained significant amounts of vitamin D2, with an average of 16.9 μg/g DM . No detC. cibarius) collected from Swedish forests had vitamin D2 between 0.12 and 6.3 μg/g DM after being hot air-dried and stored in darkness for 2–6 years [2 was most efficient when the mushroom contained 70% moisture and received UV-B radiation (in the range of 290–320 nm in this study) for 2 h, producing 25 μg D2/g DM (ca. 200 μg D2/100 g FW) [2 decreased with the moisture content dropping from 70% to 30%, although vitamin D2 concentrations were still nutritionally significant at 30% moisture content . Mushroom collectors often pick mushrooms in the wild, dry them using a hot air method, then store the dried mushrooms for months or years. When mimicking this process, chanterelles (–6 years . In a st–6 years . When sh00 g FW) . As the 2 after exposure to UV-B radiation than hot air-dried mushrooms [2 content before irradiation, freeze-dried oyster mushrooms generated 34.6 μg D2/g DM, shiitake 60 μg D2/g DM, and button mushrooms 119 μg D2/g DM after 30 min of exposure to radiation. Hot air-dried mushrooms with a moisture content of 6–8.3% produced 32–81 μg D2/g DM over the same time frame. Mushrooms that have been freeze-dried will have close to zero moisture, resulting in 8–10% of the weight of the original mushroom . Freeze-dried button, shiitake, and oyster mushrooms generated more vitamin Dushrooms . The aut2 production in button mushrooms that are freeze-dried and then powdered. For example, the ideal conditions for generating vitamin D2 from button mushroom powder were achieved by using a UV-B lamp (range 280–360 nm) with an irradiance of 1.36 W/m2 for 10 min at a temperature of 26 °C, producing 740 μg D2/g powder [2 generated was 37 μg/g DM, while 60 pulses generated 62 μg/g DM [Different variables  can influence vitamin Dg powder . When fr μg/g DM . 2 in both fresh and dried mushrooms exposed to UV radiation has mainly been done after refrigeration at 2–4 °C. Fresh button mushrooms stored at 2.2 °C showed a first-order kinetics decline in vitamin D2 concentration, with a predicted decline to a concentration of 1.75 μg/g DM at 14 days [2 concentration in sliced button mushrooms dropped from 12 μg/g DM to 8–9 μg/g DM after 3–11 days storage at 3 °C [2 concentrations in the first 24 h of storage, before vitamin D2 level gradually reduced, over 10 days, to about one third to a half of the highest post UV-exposure level [2 concentration in button mushrooms gradually increased from 3.5 μg/g DM to 8.1 μg/g DM during storage at 4 °C for six days, before dropping on days 7 and 8, while, for oyster and shiitake, vitamin D2 concentration dropped gradually over 10 days [2 losses when mushrooms were refrigerated. There was virtually no degradation of vitamin D2 when button mushrooms were refrigerated at 4 °C for 8 days [2 were found after one and four days in button mushrooms stored at 2.2 °C, equivalent to 70 μg D2/100 g FW [2 when refrigerated for one week or less.Analysis of the retention of vitamin D 14 days . The vit at 3 °C . Oyster re level . In one  10 days . Howeverr 8 days , or for r 8 days . Similar100 g FW . Conside2 up to eight months when stored in dry, dark conditions at 20 °C in closed plastic containers [2 during storage between 8 and 18 months. In the case of hot air-dried button mushrooms, vitamin D2 concentration decreased from 14.3 μg/g DM to 9.3 μg/g DM over eight months, then to 6.9 μg/g DM over the following 10 months. Three types of mushroom  exposed to a UV-B lamp and then hot air-dried, had relatively good retention of vitamin Dntainers . However2 in vitamin D-enhanced mushrooms. Following 5 minutes of frying without oil, two types of wild chanterelle mushrooms retained at least 85% of their raw-state content of vitamin D2 concentrations after adjusting for water loss during cooking [2 content of 19 μg/100 g FW, the retention of vitamin D2 after boiling in water for 20 min or oven-baking for 10 min, was 62–67%; for mushrooms pan-fried without oil for 5 min, the retention was again high, corresponding to 88% [2 retention in mushrooms.Very few studies have investigated the effect of cooking on the concentration of vitamin D cooking ,61. In b2 was from wild chanterelles in the 1990s [2 from mushrooms increased serum 25(OH)D concentrations as effectively as a vitamin D2 supplement after three weeks. Since then, the bioavailability of vitamin D2 from mushrooms has been demonstrated in both rats [2 from mushrooms supports bone health in animal models [One of the earliest studies to determine the bioavailability of vitamin Dhe 1990s . In 27 poth rats ,63,64 anoth rats ,66,67,68l models ,64,69,702 from mushrooms was assessed in a study of 30 healthy adults who were randomised to receive 2000 IU (50 μg) of supplemental vitamin D2, mushroom vitamin D2, or vitamin D3 for three months [2 from mushrooms was as effective as supplemental vitamin D2 in raising and maintaining serum 25(OH)D2 concentrations. Similarly, a five-week study in adults with serum 25(OH)D (combined 25(OH)D2 and 25(OH)D3) concentrations less than 50 nmol/L showed that vitamin D2 from soup made from UV-B irradiated mushrooms improved vitamin D status as effectively as supplemental vitamin D2 [2 concentrations in participants, whereas serum 25(OH)D3 concentrations decreased. Overall, there was no effect of the UV-irradiated mushrooms on vitamin D status [3 is more effective than vitamin D2 in raising the concentration of circulating 25(OH)D [3 is not suited to many vegetarians and that a source of vitamin D2 may be preferred.The bioavailability of vitamin De months . Vitamintamin D2 . HoweverD status . Althoug 25(OH)D ,72, it s2/100 g FW; therefore, a 100 g serve would provide 50–100% of the daily required vitamin D to consumers. Exposing dried mushrooms to UV-B radiation can also generate nutritionally useful amounts of vitamin D2, although this practice is not widespread to date.Mushroom consumption is increasing rapidly worldwide, with the production of mushrooms rising from 1 billion kg in 1978 to 27 billion kg in 2012 (an increase in per capita consumption from 0.25 kg to 4 kg) . Since m2 concentrations to nutritional significance, although pulsed UV lamps may be the most cost-efficient method for commercial production of vitamin D-enhanced mushrooms, because of the low exposure time (often in 1–3 seconds) to achieve at least 10 μg/100 g FW. There is minimal discolouration in mushrooms after pulsed UV treatment, possibly due to the small exposure time of less than 4 seconds [It is conceivable that UV-B radiation post-harvest (for fresh mushrooms) or post-drying (for dried and powdered mushrooms) could become standard commercial practice. Sunlight, regular UV lamps, and pulsed UV lamps have the capability to raise the vitamin D seconds ; however seconds ,34,36,442, which is bioavailable and relatively stable during storage and cooking. Therefore, consumption of vitamin D-enhanced mushrooms could substantially contribute to alleviating the global public health issue of vitamin D deficiency. Further research is warranted to determine the optimal level of UV radiation required to produce a nutritionally useful amount of vitamin D2 in mushrooms, along with optimal storage conditions and cooking methods. The physiological benefits of mushroom-derived vitamin D2 compared with solar-derived vitamin D3 also require further investigation.Vitamin D-enhanced mushrooms contain high concentrations of vitamin D