Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism. The industrial production of useful substances from lignin is desired, since it is the most abundant aromatic resource and the second most abundant bioresource on Earth after cellulose. In recent years, microbial conversion processes known as “biological funneling” have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained through chemical lignin depolymerization into platform chemicals5. Based on these facts, elucidating the bacterial lignin catabolic system has become more critical.Lignin is one of the major components of plant cell walls, accounting for 15–40%7. Although lignin has asymmetric carbons, it is considered optically inactive because of its equal amounts of optical isomers8. Among the intermonomer linkages in lignin, β-aryl ether is the most abundant, comprising 45%–50% of all linkages in softwood lignin and 60–62% in hardwood lignin9. Accordingly, cleavage of the β-aryl ether linkage is considered a crucial step in lignin biodegradation. Furthermore, β-aryl ether-type biaryls have two distinct isomeric forms, erythro and threo, and each form has enantiomeric forms11.Lignin is formed through the dehydrogenative polymerization of hydroxycinnamic alcohols, resulting in various C–C and C–O–C bonds between the phenylpropane unitsSphingobium sp. strain SYK-6 is an alphaproteobacterium that exhibits the best-characterized catabolic system for lignin-derived aromatic compounds12. SYK-6 can utilize lignin-derived biaryls, including β-aryl ether, phenylcoumaran, biphenyl, and diarylpropane, and monoaryls, including ferulate, vanillin, and syringaldehyde, as its sole sources of carbon and energy. In SYK-6 cells, four stereoisomers of a model β-aryl ether compound, guaiacylglycerol-β-guaiacyl ether (GGE), are converted into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through the oxidation of the α-carbon, which is catalyzed by Cα-dehydrogenases, LigD, LigL, and LigN -GGE and -GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, whereas LigL/LigN converts -GGE and -GGE into (βR)-MPHPV and (βS)-MPHPV, respectively. The ether linkage in the resulting MPHPV is cleaved by enantioselective glutathione (GSH) S-transferases (GSTs), LigF, LigE, and LigP (β-etherases), to produce α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol through the nucleophilic attack of GSH on the MPHPV β-carbon atom -MPHPV and (βR)-MPHPV, subsequently producing (βR)-GS-HPV and (βS)-GS-HPV, respectively18. Recently, X-ray crystal structures of LigE and LigF were determined, suggesting that LigE was most similar to the GSTFuA fungal class, and LigF was classified in a new structural class closely related to GSTFuAs or fungal Ure2p-like GST19. LigF and LigE cleave the β-aryl ether linkage through an SN2 nucleophilic attack of GSH on the β-carbon of the substrates19. Another GST, LigG, catalyzes the cleavage of the thioether linkage in (βR)-GS-HPV by transferring the (βR)-GS-HPV GSH moiety to another GSH molecule, producing achiral β-hydroxypropiovanillone (HPV) and oxidized glutathione (GSSG)16. Meux et al. and Pereira et al. independently solved the X-ray crystal structure of LigG21. LigG belongs to the omega-class GST, and Cys15 is a catalytic residue20. LigG had little to no activity for (βS)-GS-HPV, suggesting the involvement of an alternative GST in (βS)-GS-HPV conversion14. A recent study reported that a Nu-class GST, NmGST3 of Novosphingobium sp. strain MBES04, exhibited GSH-removing activity toward both GS-HPV isomers23. Kontur et al. reported that NaGSTNu, belonging to the Nu-class GST, showed activity toward both GS-HPV isomers, and it was the only enzyme essential for converting both GS-HPV isomers in Novosphingobium aromaticivorans DSM 1244424. Furthermore, they showed that the gene product of SLG_04120, a putative Nu-class GST in SYK-6, had GSH-removing activity for both GS-HPV isomers.In this study, we identified the GS-HPV-converting GST genes in SYK-6 and uncovered the roles of these genes in β-aryl ether catabolism, since the genes involved in GS-HPV catabolism have not been clarified in SYK-6 to date.14. High-performance liquid chromatography (HPLC) analysis of the reaction mixtures showed that MPHPV was converted into GS-HPV by purified LigF and LigE at 60 min . The conversion of MPHPV by LigF and LigE stopped at approximately 50%, and approximately equimolar GS-HPV to the converted MPHPV was generated . Chiral HPLC analysis was conducted to confirm whether LigF and LigE enantioselectively convert MPHPV. The MPHPV preparation that was used as the substrate was an equimolar mixture of (βR)-MPHPV and (βS)-MPHPV (53:47) . LigF and LigE completely converted (βS)-MPHPV and (βR)-MPHPV, respectively, indicating (βR)-GS-HPV and (βS)-GS-HPV generation in each reaction . We attempted to remove the remaining MPHPV and guaiacol from the reaction mixtures by ethyl acetate extraction; however, we found that the prepared GS-HPV isomers racemized after incubation in buffer A at 30 °C for 24 h (data not shown). Hence, we prepared each GS-HPV isomer by incubating 400 µM MPHPV with purified LigF or LigE and 5 mM GSH in buffer A before usage.Racemic MPHPV (400 µM) was reacted with purified LigF and LigE  with 5 mM GSH in 50 mM Tris–HCl buffer  to obtain enantiopure GS-HPV isomers25 containing 10 mM sucrose, 10 mM glutamate, 0.13 mM methionine, and 10 mM proline (Wx-SEMP) were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. HPLC analysis indicated that SYK-6 converted both GS-HPV isomers into HPV after incubation for 5 min . The activities of the extracts of SYK-6 cells grown in a Wx-SEMP with or without 5 mM GGE supplementation were measured to evaluate the inducibility of GS-HPV conversion. The specific activity of the extracts from the cells grown in Wx-SEMP for (βS)-GS-HPV (142 ± 3 nmol·min−1·mg−1) was approximately six-fold higher than that for (βR)-GS-HPV (24.5 ± 0.2 nmol·min−1·mg−1). The specific activities for (βS)-GS-HPV (150 ± 19 nmol·min−1·mg−1) and (βR)-GS-HPV (31.4 ± 1.1 nmol·min−1·mg−1) of the extracts from the cells grown in Wx-SEMP supplemented with 5 mM GGE were not significantly higher than those of the extracts from the cells grown in Wx-SEMP. These results suggest that GSH-removing enzyme genes are constitutively expressed.In order to verify the ability of SYK-6 to convert GS-HPV isomers, the extracts of SYK-6 cells grown in Wx minimal mediumNovosphingobium sp. strain MBES04, N. aromaticivorans DSM 12444, Novosphingobium sp. strain PP1Y, and Thiobacillus denitrificans ATCC 25259. This phylogenetic tree showed that SLG_00360 is relatively close to a clade, including ligG (19% identity), and SLG_04120 belongs to a clade, including NmGST3 of MBES04 (29% identity) and NaGSTNu of DSM 12444 (58% identity), which can convert both GS-HPV isomers. Moreover, Cys15 and Pro16, shown to be the active center of SYK-6 LigG, were conserved in SLG_29340 (Cys10 and Pro11) . On the basis of these findings, we selected ligG, SLG_00360, SLG_04120, and SLG_29340 as candidates for GS-HPV-converting GST genes. To examine whether the gene products of ligG, SLG_00360, SLG_04120, and SLG_29340 exhibit activities toward GS-HPV, these genes fused with a His-tag at the 5ʹ terminus were expressed in E. coli. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showed the protein production of 33, 28, 36, and 33 kDa, appearing as products of ligG, SLG_00360, SLG_04120, and SLG_29340 . The cell extracts of these E. coli transformants were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. HPLC analysis showed that ligG gene product completely converted (βR)-GS-HPV into HPV after incubation for 30 min, whereas it only slightly converted (βS)-GS-HPV -GS-HPV, which is similar to that of previous reports26. By contrast, the SLG_04120 gene product completely converted both GS-HPV isomers into HPV, verifying its specificity, as previously reported , ligQ mutant (ΔligQ), and ligG ligQ double mutant (ΔligG ligQ)  to determine whether ligG and ligQ are indeed involved in converting GS-HPV in SYK-6. The cell extract of each mutant was reacted with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A, and their ability to convert GS-HPV isomers was compared with SYK-6. ΔligG and ΔligQ both showed a decreased conversion of (βR)-GS-HPV, but only ΔligQ exhibited the loss of (βS)-GS-HPV conversion  and ligQ (pQFligQ) to confirm whether this activity defect was due to the disruption of ligG and ligQ. A cell extract of ΔligG ligQ harboring pQFligG showed higher specific activity for (βR)-GS-HPV than that of the wild type -GS-HPV as the wild type -GS-HPV than the wild type -GS-HPV in SYK-6 cells, whereas only ligQ is involved in (βS)-GS-HPV conversion.We then created E. coli expressing His-tagged ligG and ligQ, respectively . Purified LigG and LigQ were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. LigG showed approximately 300-fold higher specific activity for (βR)-GS-HPV (33 ± 1 µmol·min−1·mg−1) than for (βS)-GS-HPV (0.11 ± 0.01 µmol·min−1·mg−1). By contrast, LigQ showed approximately six-fold higher specific activity for (βS)-GS-HPV (110 ± 14 µmol·min−1·mg−1) than for (βR)-GS-HPV (17 ± 2 µmol·min−1·mg−1).The specific activity of LigG for (βR)-GS-HPV was approximately two-fold higher than that of LigQ. These results indicated that LigG is highly specific for (βR)-GS-HPV, and LigQ prefers (βS)-GS-HPV more than (βR)-GS-HPV. The Km values of LigG and LigQ for GSH were determined to be 1.19 ± 0.22 and 0.34 ± 0.10 mM, respectively .LigG and LigQ were purified to near-homogeneity through nickel affinity chromatography from the cell extracts of ligG and ligQ in SYK-6 affects MPHPV conversion. Here ΔligG and ΔligQ cells converted MPHPV similar to the wild type, whereas ΔligG ligQ cells lost the ability to convert MPHPV . The introduction of pQFligG or pQFligQ into ΔligG ligQ cells restored their MPHPV-converting ability, indicating that the disruption of both ligG and ligQ led to the loss of the ability to convert MPHPV in vivo -GS-HPV, and LigQ converts both GS-HPV isomers. Our study further elucidated that both ligG and ligQ participate in converting (βR)-GS-HPV in SYK-6 cells, whereas only ligQ is involved in converting (βS)-GS-HPV -GS-HPV conversion by ΔligQ for 60 min was lower than that by ΔligG, ligQ appears to more involved in (βR)-GS-HPV conversion than ligG -GS-HPV was approximately two-fold lower than that of LigG in the presence of 2.4 mM GSH. The reason why the disruption of ligQ had more influence on (βR)-GS-HPV conversion than the disruption of ligG may be the higher GSH affinity of LigQ than that of LigG . Alternatively, ligQ expression level in SYK-6 may be higher than that of ligG.We confirmed that LigG is highly specific for  and NmGST6 (MBENS4_2530) that showed 29% identity with LigQ and 62% identity with LigG, respectively. NmGST3 had activities for both isomers, and NmGST6 was highly specific for (βR)-GS-HPV23; however, the involvement of these genes in GS-HPV catabolism has not been investigated. We found another GST gene in the MBES04 genome, MBENS4_4395 (accession no. GAM07399), exhibiting 59% and 63% identity with LigQ and NaGSTNu, respectively. GAM07399 may be involved in GS-HPV isomer conversion besides the NmGST3 and NmGST6 genes. In Novosphingobium sp. strain PP1Y, NsLigG (PP1Y_AT11674), exhibiting 63% identity with LigG, was highly specific for (βR)-GS-HPV26. Furthermore, we found PP1Y_AT11650 (accession no. CCA92086) in the genome of this strain, which showed 80% identity with NmGST3; and PP1Y_AT20084 (accession no. CCA92884) that showed 57% and 66% identity with LigQ and NaGSTNu, respectively. These genes may also be involved in GS-HPV isomer conversion.In ligD, ligF, ligE, and ligG, constitute an operon, whereas ligQ locates at a different locus 14. In the case of MBES04, the NmGST4–NmGST5–NmGST6 genes (MBENS4_2528–2530) corresponding to ligFEG are adjacent, and the NmGST3 gene  locates just upstream of MBENS4_2528 with a different transcription direction 22. By contrast, another Nu-class GST gene MBENS4_4395 (accession no. GAM07399) locates at a different locus. Almost the same gene organization was also observed in the PP1Y genome 22. Based on the conservation of these gene arrangements, the conversion of both GS-HPV isomers may become possible by acquiring a Nu-class GST gene in strains carrying ligFEG or by adding ligFEG to a Nu-class GST gene-carrying strain. Unlike the aforementioned strains, the genes corresponding to ligDFE are scattered through the DSM 12444 genome.In the SYK-6 genome, the β-aryl ether catabolic genes, S)-GS-HPV (0.11 ± 0.01 µmol·min−1·mg−1) was only 0.33% of that for (βR)-GS-HPV (33 ± 1 μmol·min−1·mg−1). This result is similar to previous reports demonstrating that LigG had little to no activity for (βS)-GS-HPV derivatives26. By contrast, Rosini et al. reported that 500 µM racemic GGE was fully converted into HPV and guaiacol by purified LigD, LigL, LigF, LigE, and LigG in 2 h27, suggesting that LigG catalyzed the thioether cleavage of both (βS)- and (βR)-GS-HPV. However, the conversion of (βS)-GS-HPV by LigG was likely to have proceeded because a large amount of purified LigG (200 µg/ml) was used for this conversion.The specific activity of LigG for (βS)-GS-HPV than for (βR)-GS-HPV in this study. Kontur et al. reported that LigQ showed approximately 11-fold higher kcat/Km value for (βS)-GS-HPV than for (βR)-GS-HPV (Table S2)24. Also, NaGSTNu of DSM 12444 showed approximately four-fold higher kcat/Km value for (βS)-GS-HPV than for (βR)-GS-HPV24, and NmGST3 of MBES04 had approximately two-fold higher specific activity for (βS)-GS-HPV than for (βR)-GS-HPV (Table S2)23. Therefore, all these Nu-class GSTs have higher specificity for (βS)-GS-HPV than for (βR)-GS-HPV.Conversely, LigQ showed approximately six-fold higher specific activity for . These values were considerably higher than Km of LigG for (βR)-GS-HPV (0.016 ± 0.001 mM)21 and Km of LigQ for (βR)-GS-HPV (0.055 ± 0.007 mM) and (βS)-GS-HPV (0.011 ± 0.002 mM)24. The Km value of LigG for GSH is supported by the observation of Picart et al. that the specific activity of LigG was halved when reacted in the presence of 0.4 mM GSH compared to 1 mM GSH26. Based on the quantum mechanics/molecular mechanics simulations, Prates et al. suggested that HPV leaves the active site after GS– of GS-HPV is transferred to Cys15 of LigG, giving enough space to accommodate a second GSH for a subsequent thiol-disulfide reaction, which forms GSH disulfide and restores the catalytic ability of LigG18. The lack of specific interactions to bind the GSH in the active site of LigG was suggested to be associated with high Km value for GSH estimated by the results of Picart et al18. In addition, the Km values for GSH of E. coli Nu-class GSTs (YghU and YfcG) have been reported to be on the order of mM when using 2-hydroxyethyl disulfide as a substrate 29.The Nu was proposed based on X-ray crystal structure, modeling of substrate binding, and mutant analysis. Thr51 and Asn53 of NaGSTNu provide hydrogen bonds that stabilize a reactive GSH thiolate anion, which attacks the GS− moiety of GS-HPV to form GS–SG disulfide. The rupture of the thioether bond is facilitated by the formation of a transient enolate intermediate. This intermediate is stabilized by the interactions between GS-HPV and Tyr166 and Tyr224 hydroxy groups. The capture of a solvent-derived proton by the carbanion collapses the enolate to form HPV24. All these residues are conserved in LigQ, GAM07399, and CCA92884, suggesting that these GSTs catalyze GSH removal from GS-HPV using the same reaction mechanism as that of NaGSTNu.The mechanism for the cleavage of the thioether bond in GS-HPV by NaGSTligG ligQ lost the ability to convert MPHPV in vivo  could not proceed , Wx-SEMP, and Wx-SEMP containing 5 mM GGE at 30 °C. When necessary, 50 mg kanamycin/liter, 100 mg streptomycin/liter, or 12.5 mg tetracycline/liter were added to the cultures. E. coli strains were grown in LB at 37 °C. The media for E. coli transformants were supplemented with 100 mg ampicillin/liter, 25 mg kanamycin/liter, or 12.5 mg tetracycline/liter.Table S3 lists the strains and plasmids that were used in this study. 31. In preparing (βR)-GS-HPV and (βS)-GS-HPV, purified LigF (40 µg protein/ml) and LigE (150 µg protein/ml) were incubated in 1 ml reaction mixtures containing 400 µM racemic MPHPV, 5 mM GSH, and buffer A, respectively, for 60 min at 30 °C14. The resulting mixtures were used as preparations of 200 µM GS-HPV isomers, including 4.8 mM GSH. GGE and guaiacol were purchased from Tokyo Chemical Industry, Co., Ltd.MPHPV and HPV were prepared as described previouslyHPLC analysis was conducted using the ACQUITY UPLC system (Waters). The MPHPV and GS-HPV reaction products were analyzed using a TSKgel ODS-140HTP column . Additionally, a CHIRALPAK IE column  was used to analyze MPHPV reaction products. All analyses were conducted at a flow rate of 0.5 ml/min. The mobile phase was a mixture of solution A (acetonitrile containing 0.1% formic acid) and B (water containing 0.1% formic acid) under the following conditions: detection of MPHPV reaction products using TSKgel ODS-140HTP: 0–2.0 min, 10% A; 2.0–4.0 min, linear gradient from 10 to 60% A; 4.0–4.5 min, linear gradient from 60 to 90% A; 4.5–5.0 min, 90% A. Detection of MPHPV reaction products using CHIRALPAK IE: 0–25.0 min, 30% A. Detection of GS-HPV reaction products using TSKgel ODS-140HTP: 0–5.0 min, 10% A. MPHPV, GS-HPV, guaiacol, and HPV were detected at 280, 286, 276, and 276 nm, respectively.ligG, ΔligQ, and ΔligG ligQ) grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an optical density at 600 nm (OD600) of 0.2, and incubated for 16 h. The resultant cells were washed twice with buffer A. The cells that were resuspended in the same buffer were then broken by an ultrasonic disintegrator , and the supernatants were obtained as cell extracts after centrifugation . Protein concentration was determined using the Bradford method, using bovine serum albumin as the standard (Bio-Rad Laboratories).GS-HPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring HPV production using HPLC. The MPHPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring the decrease in MPHPV using HPLC. The reaction products were also detected using HPLC analysis. SYK-6 and its mutant cells (ΔR)-GS-HPV, (βS)-GS-HPV, or 200 µM racemic MPHPV in the presence of 2.4 mM (for GS-HPV) or 5.0 mM GSH (for MPHPV) in buffer A at 30 °C. The reactions were stopped by adding acetonitrile  after 3 min (measurement of the specific activities for GS-HPV), 5 min (identification of GS-HPV metabolites), 20 min (measurement of the specific activities for GS-HPV in mutant-complemented strains), or 60 min (for MPHPV). Precipitated proteins were removed by centrifugation at 19,000 × g for 15 min, and the resulting supernatants were diluted with water to a final acetonitrile concentration of 12.5%, filtered, and analyzed using HPLC. The specific activity of GS-HPV conversion was expressed in moles of HPV produced per min per milligram of protein. The cells of SYK-6 grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an OD600 of 0.2, and incubated at 30 °C to examine the effects of GGE on enzyme induction. When the cultures reached an OD600 of 0.4–0.5, we added 5 mM GGE to them. After 12 h of further incubation, cell extracts were prepared and used for the enzyme assay. For complementing ΔligG ligQ, pQFligG and pQFligQ were constructed by In-Fusion cloning (Takara Bio) of ligG and ligQ fragments amplified by PCR using SYK-6 total DNA and their corresponding primer pairs (Table S4) into pQF. After confirming the nucleotide sequences of the inserts, each plasmid was introduced into ΔligG ligQ cells through electroporation. The transformed cells were grown in Wx-SEMP containing 100 µM cumate and tetracycline for 16 h, and the cell extracts were prepared and used for the enzyme assay.Cell extracts (5–1000 µg protein/ml) were incubated with 100 µM , applying 1,000 bootstrap replicates.Nucleotide sequences were determined through Eurofins Genomics. Sequence analysis was conducted using the MacVector program . Sequence similarity searches, pairwise alignments, and multiple alignments were conducted using the BLASTP programligQ (SLG_04120), ligF (SLG_08650), ligE (SLG_08660), ligG (SLG_08670), and SLG_29340 were amplified through PCR using SYK-6 total DNA and the primer pairs listed in Table S4, and each amplified fragment was cloned into pET-16b by In-Fusion Cloning. The nucleotide sequences of the inserts were confirmed by sequencing. Each expression plasmid was introduced into E. coli BL21(DE3), and the transformed cells were grown in LB. Gene expression was induced for 4 h at 30 °C by adding 1 mM isopropyl-β-D-thiogalactopyranoside when the OD600 of the cultures reached 0.5. The cells were then harvested by centrifugation at 5000×g for 5 min at 4 °C and washed with buffer A. The cells were then resuspended in the same buffer and broken using an ultrasonic disintegrator. The supernatants were obtained as cell extracts after centrifugation at 19,000×g for 15 min at 4 °C. For purification of LigF, LigE, LigG, and LigQ, the cell extracts of E. coli BL21(DE3) harboring pET08650, pET08660, pET08670, and pET04120, respectively (Table S3), were transferred onto a His SpinTrap column . The purified fractions were subjected to desalting and concentration using an Amicon Ultra centrifugal filter unit , and the enzyme preparations were stored at − 80 °C. Gene expressions and the purity of the preparations were examined using SDS-12% PAGE. The protein bands in gels were stained with Coomassie Brilliant Blue.DNA fragments carrying SLG_00360, R)- and (βS)-GS-HPV, respectively; LigQ, 0.2 and 1.0 µg protein/ml for the conversion of (βS)- and (βR)-GS-HPV, respectively) were incubated in the 100 µl reaction mixtures containing 100 µM (βR)-GS-HPV or (βS)-GS-HPV, 2.4 mM GSH, and buffer A at 30 °C. The reactions were stopped by adding acetonitrile or methanol to a final concentration of 50% after 30 min of incubation (for cell extracts) and 15 s (for purified enzymes). Protein precipitates were removed through centrifugation at 19,000×g for 15 min. The resulting supernatants were diluted with water to a final concentration of acetonitrile or methanol of 12.5%, filtered, and analyzed using HPLC. Specific activities for GS-HPV conversion of purified enzymes were calculated after 15 s incubation and expressed in moles of HPV produced per min per milligram of protein. The Km values of LigG and LigQ for GSH were determined using 100 µM (βR)-GS-HPV under the following protein and GSH concentrations: LigG, 1.25 µg/ml protein and 0.25–4.9 mM GSH; LigQ, 5.0 µg/ml protein and 0.1–1.9 mM GSH. The Km values were calculated through non-linear regression analysis using the GraphPad Prism 7 software  fitted to the Michaelis–Menten equation and expressed as means ± standard deviations of three independent experiments.GS-HPV-transforming activities were determined by measuring HPV production using HPLC. The reaction products were also detected through HPLC analysis. The cell extracts (100 µg protein/ml) and purified enzymes  were incubated at 30 °C with shaking for 24 h. Portions of the cultures were periodically collected, and the amounts of the MPHPV were measured using HPLC. For complementation analysis of ΔligG ligQ, the resting cells (OD600 of 5.0) of SYK-6 harboring pQF, ΔligG ligQ harboring pQF, ΔligG ligQ harboring pQFligG, or ΔligG ligQ harboring pQFligQ, grown in Wx-SEMP containing cumate (100 µM) and tetracycline, were incubated with 200 µM MPHPV at 30 °C with shaking for 24 h.The cells of SYK-6, ΔSupplementary information.


JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.Misfolding mutations in cartilage oligomeric matrix protein (COMP) cause it to be retained within the endoplasmic reticulum (ER) of chondrocytes, stimulating a multitude of damaging cellular responses including ER stress, inflammation, and oxidative stress, which ultimately culminates in the death of growth plate chondrocytes and pseudoachondroplasia (PSACH). Previously, we demonstrated that an antioxidant, resveratrol, substantially reduces the intracellular accumulation of mutant‐COMP, dampens cellular stress, and lowers the level of growth plate chondrocyte death. In addition, we showed that resveratrol reduces mammalian target of rapamycin complex 1 (mTORC1) signaling, suggesting a potential mechanism. In this work, we investigate the role of autophagy in treatment of COMPopathies. In cultured chondrocytes expressing wild‐type COMP or mutant‐COMP, resveratrol significantly increased the number of Microtubule‐associated protein 1A/1B‐light chain 3 (LC3) vesicles, directly demonstrating that resveratrol‐stimulated autophagy is an important component of the resveratrol‐driven mechanism responsible for the degradation of mutant‐COMP. Moreover, pharmacological inhibitors of autophagy suppressed degradation of mutant‐COMP in our established mouse model of PSACH. In contrast, blockage of the proteasome did not substantially alter resveratrol clearance of mutant‐COMP from growth plate chondrocytes. Mechanistically, resveratrol increased SIRT1 and PP2A expression and reduced MID1 expression and activation of phosphorylated protein kinase B (pAKT) and mTORC1 signaling in growth plate chondrocytes, allowing clearance of mutant‐COMP by autophagy. Importantly, we show that optimal reduction in growth plate pathology, including decreased mutant‐COMP retention, decreased mTORC1 signaling, and restoration of chondrocyte proliferation was attained when treatment was initiated between birth to 1 week of age in MT‐COMP mice, translating to birth to approximately 2 years of age in children with PSACH. These results clearly demonstrate that resveratrol stimulates clearance of mutant‐COMP by an autophagy‐centric mechanism. © 2020 The Authors.  This is particularly true for skeletal dysplasias, in which 425 distinct disorders are caused by mutations in 437 different genes. Mouse modeling of the genetic mutations generating specific skeletal dysplasia has revealed the mechanistic pathways that underlie the resulting pathologies, thereby identifying therapeutic targets. Previously, we showed that mutations in cartilage oligomeric matrix protein (COMP) causes pseudoachondroplasia , a well‐recognized and clinically characterized severe dwarfing condition. The MT‐COMP transgenic mouse, with the common D469del mutation that recapitulates the short stature and skeletal finding of PSACH, was used to define the pathology that specifically affects growth plate chondrocytes. In this study, mechanistic‐driven therapeutic approaches in the MT‐COMP mouse were used to define the mechanisms of resveratrol therapy.Next‐generation sequencing has resulted in a huge expansion of information that has led to greater understanding of the mutational underpinnings of genetic disorders.1 This It has long been known that mutant protein accumulates in PSACH chondrocytes; mutations in COMP were shown to cause PSACH. It was subsequently shown that mutant‐COMP misfolds in the endoplasmic reticulum (ER) and prematurely assembles an ordered matrix involving collagens type 2 and 9, matrilin 3, and other extracellular proteins, resulting in massive intracellular protein accumulation. The protein accumulation in the ER of chondrocytes initiates a self‐perpetuating pathological loop between oxidative stress, inflammation, and ER stress that leads to elevated mammalian target of rapamycin complex 1 (mTORC1) signaling, DNA damage, and death of chondrocytes. Misfolded proteins in the ER are typically degraded through macroautophagy (henceforth referred to as autophagy) or by the proteasome after being translocated out of the ER. In the presence of mutant‐COMP, the high volume of stalled protein/misfolded protein/intracellular matrix either overwhelms the cellular clearance mechanisms and/or blocks export and degradation. In either situation, these clearance mechanisms do not efficiently clear mutant‐COMP from the ER and normal chondrocyte function is disrupted.Chondrocytes are secretory cells that generate abundant extracellular matrix (ECM) proteins necessary to build cartilage. COMP is normally synthesized in the rough endoplasmic reticulum (rER) cisternae and is processed through the Golgi then exported to the ECM where it forms an ordered matrix network.8 It h Notably, these treatments rescued 50% of the loss of femur length. These studies now define the cellular processes stimulated by resveratrol (aspirin) that prevent mutant‐COMP accumulation and, importantly, define the appropriate timing of treatment.Previously, we demonstrated that treatment of MT‐COMP mice with resveratrol or aspirin from birth to 4 weeks decreased mutant‐COMP intracellular retention, inflammation, and chondrocyte cell death, and restored chondrocyte proliferation.14 NotMus musculus) mice used in these and previously described experiments contain the pTRE‐COMP (coding sequence of human COMP+FLAG tag driven by the tetracycline responsive element promoter) and pTET‐On‐Col II (reverse tetracycline transactivator [rtTA] coding sequence driven by a type II collagen promoter). The presence of these two transgenes had not led to any readily detectable health concerns. Male and female mice were administered doxycycline (DOX) (500 ng/mL) postnatally in their drinking water until collection. Mice were housed as a single‐sex group after weaning and fed standard chow . Ten mice were in each treatment group or control group and litters were assigned to each group until each group contained at least 10 animals. Investigators were not blinded during allocation and animal handling but were blinded at analysis. These studies were approved by the UTHealth Animal Welfare Committee.The MT‐COMP (C57BL/6 romoter).15 The The limbs were fixed in 95% ethanol and pepsin (1 mg/mL in 0.1 N HCl) was used for antigen retrieval. Immunostaining was performed by incubating different sections overnight at 4°C using the following antibodies: human COMP , pS6 , PI3K‐1 , SIRT‐1 , phosphorylated adenosine monophosphate‐activated protein kinase (pAMPK) , interleukin 16 (IL‐16) , YM1/ECF‐L , and PCNA . Species‐specific biotinylated secondary antibodies were used for 1 hour at room temperature (RT). Sections were then washed and incubated with streptavidin horseradish peroxidase (HRP) and diaminobenzidine (DAB) was used as chromogen. The sections were dehydrated and mounted with Thermo Fisher cytoseal 60  and then visualized under the BX51 inverted microscope . Limbs were fixed in 10% wt/vol formalin for terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining. A priori data indicate that the percentage TUNEL of positive chondrocytes is approximately 80% in untreated mice and 20% in mice treated with resveratrol from birth to 4 weeks, with within‐group standard deviations (SDs) of around 12%. For a one‐way ANOVA analysis with post‐hoc Tukey test to identify difference in TUNEL percentages between five groups at a type I error rate of 0.05, a sample size of 10 mice per group would have 80% power to identify an effect size (Cohen's d) of 0.51. This would translate into pairwise differences in means of at least 17% in at least two of the five groups. Human COMP antibody was validated using C57 negative control mice and human chondrocyte positive control. Both positive and negative (no secondary antibody) controls are including in all experiments.Hind limbs from male MT‐COMP and C57BL\6 control mice were collected and tibial growth plates analyzed as described.16 TheNo adverse events were associated with these drugs administered as described. Rapamycin treatment at 2 mg/kg reduced viability of pups and the dosage was reduced to 1 mg/kg. was administered in a dose of 0.25 g/L in the drinking water starting either from birth  or 1, 2, or 3 weeks and continued until 4 weeks of age.Resveratrol , with known antioxidant/anti‐inflammatory properties,19 wasAspirin, a COX2 inhibitor that reduces inflammation was administered in the drinking water in a dose of 0.3 g/L of aspirin  from birth to 4 weeks.3 was dissolved in ethanol at 1 mg/mL and stored at −20°C. On the day of injection, rapamycin was diluted with PBS to 0.1 mg/mL. Intraperitoneal injection of rapamycin at 1 mg/kg was performed 5 days per week from 1 to 4 weeks of age.Rapamycin , which inhibits the mTOR pathway, inducing autophagy,20 was was administered at 0.5 mg/mL in DOX drinking water from birth  to 4 weeks.Metformin (Sigma‐Aldrich), which activates AMPK, in turn represses mTORC1 activity and stimulating autophagy,23 was was dissolved in 10 mg/mL DMSO, aliquoted, and stored at −20°C. For each subcutaneous injection, bafilomycin A was freshly diluted to 0.05 mg/mL in PBS. Mice were injected with 0.3 mg/kg of bortezomib five times per week beginning at age 1 week. Bafilomycin A administration to untreated MT‐COMP mice did not alter mutant‐COMP accumulation.Bafilomycin A , which blocks autophagy by preventing fusion of autophagosome with lysosome,25 was was dissolved in 10 mg/1 mL DMSO, aliquotted, and stored at −20°C. Freshly diluted bortezomib was diluted for each subcutaneous injection to 0.01 mg/mL in PBS. Mice were injected with 0.1 mg/kg of bortezomib three times per week beginning at age 1 week. Bortezomib administration to untreated MT‐COMP mice did not alter mutant‐COMP accumulation.Bortezomib , which inhibits the chymotryptic activity of the proteasome (20S subunit) reducing proteasomal degradation,27 washttps://imagej.nih.gov/ij/) was used to count LC3 foci on 10 images for each group with 30 to 50 cells per image; t tests were used to compare each group to the other groups, and scorers were blinded.Rat chondrosarcoma (RCS) cells expressing either human wild‐type or mutant‐COMP were generated using the Lenti‐X‐Tet‐On advanced inducible expression system according to the manufacturer's protocol . Briefly, RCS cells were infected with high‐titer lentiviral preparations of pLVX‐Tet‐on advanced vector or pLVX‐Tight‐human D469del‐MT‐COMP vector; and selected with puromycin and G418. Expression of human COMP in RCS cells was validated by Western blot with anti‐Flag antibody to recognize the tagged COMP . DOX was used for 4 days to induce COMP expression in RCS cells, which were then treated for 1 day as follows: (i) control with no chloroquine and no resveratrol; (ii) chloroquine (50μM); (iii) resveratrol (50μM); and (iv) both resveratrol (50μM) and chloroquine (50μM). The cells were fixed posttreatment with methanol at −20°C for 10 min, rinsed with PBS, and stored at 4°C. Coverslips were then incubated for 1 hour at 37°C with light chain 3 (LC3) primary antibody . Primary antibodies were detected using Alexa Fluor 594 for 1 hour at 37°C (dilution 1:1000). Images were collected with confocal microscope at 63× oil immersion lens . A priori data suggests that the median number of foci per image for each of the eight groups would range from 0 through 2 with interquartile ranges of 0 through 3. Assuming a power of 0.80 and a type I error rate of 0.05, a sample size of 23 cells counted per image will provide us with sufficient power to identify a small effect size of 0.1 (Cohen's d). For our analysis, we elected to count 30 to 50 cells per image. ImageJ software  or a proteasomal inhibitor (bortezomib) from 1 to 4 weeks of age. As shown in Fig.  The addition of bafilomycin A largely prevented resveratrol‐stimulated clearance of mutant‐COMP ‐COMP or mutant‐COMP were treated with resveratrol in presence or absence of chloroquine and the number of microtubule‐associated LC3 positive vesicles (foci), the classic marker of autophagy, were assessed.31 As  SIRT1 enhances cellular survival by deacetylating LC3, which promotes autophagy. To investigate the role of SIRT1 in resveratrol autophagy reactivation, growth plates were evaluated for the presence of SIRT1 in untreated MT‐COMP mice and those treated from birth to 4 weeks of age with resveratrol. SIRT1 was suppressed in MT‐COMP growth plate chondrocytes  inflammation and ER stress. We asked whether metformin treatment could induce autophagy in MT‐COMP mice, thereby mimicking resveratrol's mechanism of action. Here, we assessed whether mTORC1 signaling could be reduced by metformin‐stimulated AMPK activation in growth plate chondrocytes. As shown in Fig. Metformin modulates mTORC1 signaling by activating AMPK,40 whi(11) To define the treatment windows in which resveratrol could be administered to MT‐COMP mice to reduce chondrocyte pathology, resveratrol was started at birth, 1, 2, or 3 weeks of age and growth plates were collected at 4 weeks. As shown in Fig. Previously, we demonstrated that resveratrol treatment in MT‐COMP mice from birth to 4 weeks of age dampens mutant‐COMP intracellular ER retention, inflammation, chondrocyte death, and restored chondrocyte proliferation, resulting in increased femoral length. and is caused by the retention of mutant‐COMP in the ER, which stimulates a chronic self‐perpetuating pathological loop between ER stress, inflammation, and oxidative that elevates mTORC1 signaling through a complex network involving CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and TNFα  is unknown.3 in vrol Fig.  and rapa Many storage disorders involve a failure of or inefficient autophagy including proteinopathies , lipid storage disorders , and glycogen storage disorders . Autophagy failure in disease occurs through a variety of mechanisms including impaired autophagosome formation, compromised autophagosome maturation , and improper recognition of autophagic cargo. In PSACH, a COMPopathy/proteinopathy, mTORC1 activation and SIRT1 downregulation blocks autophagosome maturation through a complex network , our findings suggest that resveratrol treatment at or shortly after diagnosis will likely provide some reduction of clinical and growth plate pathology. Importantly, 3 weeks of resveratrol treatment (treatment from 1 to 4 weeks) in the MT‐COMP mouse increased autophagy , showing that autophagy reactivation is linked to the recovery of chondrocyte proliferation, which most likely accounts for the recovery of some limb growth with resveratrol treatment. Interestingly, treatment beginning as late as 3 weeks of age (treatment from 3 to 4 weeks) reduced inflammation and, therefore, would be expected to decrease joint pain, suggesting that even late treatment may have some benefit. The results for aspirin were similar to those with resveratrol with the exception that at least 2 weeks of aspirin treatment was required to reduce intracellular mutant‐COMP . Importantly, resveratrol was more effective than aspirin at reducing mutant‐COMP ER accumulation, most likely related to its effect on multiple molecules that stimulate autophagy and reduce inflammation and oxidative stress , although earlier treatment would likely be more effective based on the findings presented here.The outcome of these studies have important therapeutic applications because they show there is a therapeutic window during juvenile mouse growth in which resveratrol treatment diminished the mutant‐COMP chondrocyte pathology. This is a significant finding because most PSACH cases are not evident at birth. The absence of linear growth prompts diagnostic testing in children leading to PSACH diagnosis by 2 years of age. Moreover, these findings have important implications for addressing PSACH childhood pain that has not previously been appreciated or systematically treated.67 ResResveratrol treatment is promising given that the potential therapeutic window spans the postdiagnosis period in PSACH and resveratrol decreases multiple cellular stresses, stimulates autophagy clearance of mutant‐COMP, allowing chondrocytes to function and proliferate, potentially increasing long‐bone growth. Collectively, these findings suggest that chondrocyte death, inflammation, and pain are likely to improve prior to recovery of growth with resveratrol therapy and earlier treatment results in the best outcome.The authors have no conflict of interest to declare.Jacqueline Hecht: Funding acquisition; project administration; resources; supervision; writing‐review and editing. Francoise Coustry: Data curation; investigation; methodology; visualization. Alka Veerisetty: Data curation; investigation; methodology. Mohammad Hossain: Data curation; investigation; methodology. Karen Posey: Conceptualization; formal analysis; funding acquisition; project administration; supervision; writing‐original draft; writing‐review and editing.https://publons.com/publon/10.1002/jbm4.10456.The peer review history for this article is available at Supplementary Fig. S1. Aspirin treatment window. Treatments started at 1 or 2 or 3 weeks after birth and stopped at 4 weeks and evaluated for mutant‐COMP pathology. Each treatment group included 10 mice. Aspirin treatments are shown in the shaded boxes. Treatments starting at birth or 1 week of age produced the best therapeutic outcomes , later therapy mitigated but did not eliminate the disease progression (C). Control (C57BL\6)  and MT‐COMP with no treatment  resveratrol treatment for 1 , 2 , 3  or 4  weeks and growth plates at 4 weeks were evaluated for human COMP retention, IL‐16 and YM1, TUNEL (cell death), PCNA and mTORC1 signaling. Quantification of TUNEL positive chondrocytes for different treatment periods are shown in KK. Bars = means with standard deviation. Aspirin treatment decreased intracellular MT‐COMP after 2 weeks of treatment (D) and decreased in IL‐16 and YM1 inflammation after 1 week of treatment  compared to . Aspirin decreased cell death after 2 weeks of treatment (U), mTORC1 signaling after 3 weeks of treatment (JJ) and increased proliferation after 3 weeks of treatment (CC). The relative change of MT‐COMP pathology on treatment period is shown in LL.Click here for additional data file.Supplementary Fig. S2. Rapamycin activates autophagy in MT‐COMP growth plate chondrocytes. Rapamycin reduces MT‐COMP retention in chondrocytes. Immunostaining with human COMP antibody of: (A) MT‐COMP growth plate chondrocytes at 4 weeks with no treatment, (B) control vehicle, (C) rapamycin (1 mg/kg 5 i.p. injections per week from 1 to 4 weeks of age). Ten mice were included in each treatment group.Click here for additional data file.


Three human papillomavirus (HPV) vaccines are available against up to nine HPV types. In Sweden, from 2012, Gardasil was offered to 10−12 year old girls through the school-based vaccination program, and as catchup vaccination for women up to 26 years. To obtain a baseline, and follow HPV vaccination effects, during 2008−2018, cervical and oral HPV prevalence were followed at a youth clinic in Stockholm, and in 2013 for comparison oral HPV prevalence was examined in high-school youth in a middle-sized county in Sweden. In this review, we discuss all our data with cervical and oral mouthwash samples that were collected and tested for 24−27 HPV types by a bead-based multiplex assay from 2008. Compared with 2008−2011, with ~ 35% HPV16 and > 60% high risk (HR) HPV cervical prevalence at the youth clinic, a decrease of vaccine HPV types was observed between 2013 and 2018, with e.g., HPV16 falling to 5% in catchup vaccinated women and 15−18% in nonvaccinated women. Most common cervical HR-HPV types were HPV39, 51, 52, 56, and 59 together accounting for ~ 10% of cervical cancer, and where only HPV52 is included in Gardasil-9. At baseline 2009−2011, oral HPV prevalence was ~ 10% in unvaccinated youth at the youth clinic, but after 2013 it dropped to < 2% at the youth clinic and high schools.To conclude, Gardasil HPV types have decreased, but it is still important to follow remaining HR-HPV types and cancer development, since there is an ongoing increase in the incidence of HPV-associated tonsillar and base of tongue cancer, and cervical cancer in Sweden. In 1983, when HPV16 was finally disclosed in cervical cancer, this finding was complex since several HPV types were being discovered and found to cause cervical cancer .Today, > 200 HPV types have been discovered, of which most are cutaneous and ~ 40 mucosal and defined as low risk (LR), putatively high risk (HR), or HR−HPV types . All havIn 1995, HPV16 was recognized as a carcinogen in cervical cancer by the International Agency for Research against cancer (IARC) –10 By thIn parallel, in 2006, the first HPV vaccine Gardasil, which against HPV16, 18, 6 and 11, was approved by the Food and Drug Administration. This was the case for Cervarix (against HPV16 and 18) and Gardasil-9  in 2009 and 2014, respectively .This review presents details of the period during the introduction of the HPV vaccine with focus on young individuals at a youth clinic in Stockholm, Sweden, but also in youth at high schools in a median-sized county in Sweden Table . In addiHPV vaccination was gradually introduced in Sweden between 2006−2011. In 2012, Gardasil was supplied for 10−12 year old girls through the school-based vaccination program, and catchup vaccination of girls aged 13−26 years was also offered for free, depending on Swedish county .–20.18–20Oral HPV prevalence also dropped considerably to 1.5% (7/457) when assessed at the youth clinic in the study of 2013−2015 (Table To conclude, at the youth clinic both cervical and oral HPV prevalence were fairly high from an international perspective, and in comparison to the relatively few reports that existed during similar periods , 17, 24.In a recent systemic review and meta-analysis, the data from 60 million individuals were covered in 65 articles from 14 high-income countries, and of these 65 articles, 23 followed HPV infection, 29 followed anogenital warts, and 13 followed CIN2 + . The metScreening of cervical cancer is according to the National Board of Health and Welfare today recommended, by cytology for women 23−29 years every third year, by HPV testing for women 30−49 years every 3rd year, and by complementary cytology at the age of 41 years . For womAs mentioned above, TSCC and BOTSCC are increasing in incidence, and possible screening has been discussed. However, the presence of oral HPV infection is relatively rare, likely due to the secretion of saliva in the oral cavity. Furthermore, others and we have shown that the presence of HPV in the oral cavity is not necessarily associated with the presence of HPV in the tonsils, and therefore not necessarily correlated to cancer development . MoreoveNonetheless, we have shown that patients with HPV-positive TSCC and BOTSCC often have high MFI levels of the tumour HPV type in their oral cavity upon donating a gargle sample, and these levels are roughly 200 times higher than the HPV MFIs in oral rinses of healthy youth , 29. It Notably, recently attempts have also been made to look at pre-stages of TSCC and BOTSCC, and we have also compared dysplastic lesions in TSCC to invasive cancer . Forty gLactobacilli species or Lactobacillus iners were associated with three to five times higher odds of any prevalent HPV and two to three times higher for high-risk HPV and dysplasia/cervical cancer compared with Lactobacillus crispatus [Vaginal microbiota has recently been linked to an increased risk of sexually transmitted infections, including HPV infection . Our datrispatus . Meta-anrispatus . A potenTo summarize, presentation of HPV vaccination in the school-based program and as catchup vaccination has successfully decreased the cervical and oral HPV prevalence of the Gardasil HPV16, 18, 6, and 11 types, both in vaccinated and nonvaccinated youth. Continued HPV vaccination on both genders would be extremely valuable and allow for a further decrease in HPV vaccine types and their associated cancers .Nonetheless, other HR-HPV types still persist, of which most are not included in Gardasil-9 and these accounted for 10% of all cervical cancer in the Stockholm region 2003−2008. Therefore, it is important to follow-up patients with cervical screening, and investigate which HR-HPVs are present in HPV-associated tumors in the future.In addition, more knowledge on microbiota may allow for better means of combating persistent HPV infections. Future cancer screening could also use the knowledge acquired on different protein expression in dysplastic and invasive lesions of TSCC and BOTSCC.


Despite a growing understanding of the importance of provider HPV recommendation on parental acceptance, U.S. HPV vaccination rates remain suboptimal. Given the prevalence and use of the media for health decisions, this study examined the relationship between the media and provider HPV recommendation on maternal HPV vaccine hesitancy.Thirty individual interviews with HPV vaccine-accepting mothers in the Midwest U.S. were conducted to examine their feelings of hesitancy around the decision to accept HPV vaccination at the time of provider recommendation and their suggestions for improving the recommendation experience by addressing media concerns.Media exposure was an antecedent to hesitancy for three main vaccination concerns: safety, protection/efficacy and sexual stigma. Although mothers accepted vaccination, they continued to feel confused and hesitant about HPV vaccination. They had several recommendations for how providers could combat hesitancy to improve confidence in HPV vaccine acceptance.Providers’ approach to HPV vaccination recommendation must consider concerns reported in the media with delivery techniques modified to adjust to maternal fears absorbed from adverse media information.The online version contains supplementary material available at 10.1186/s12889-020-09877-x. It is well known that the human papillomavirus (HPV) causes nearly all cervical cancers and many cancers of the vagina, vulva, penis, anus, and oropharynx . In the The poor uptake rate of the HPV vaccine has been attributed to many factors, of which parental hesitancy is one of the strongest . Many stLow coverage rates have prompted considerable research on the determinants of HPV vaccination. The primary influence on parental HPV decisions highlighted in the literature is the healthcare providers’ recommendation; those who receive a provider recommendation are more likely to vaccinate their children than those who do not , 11. YetMisinformation and hesitancy among HPV vaccine accepting parents are largely unexplored. In today’s health conscious society, seeking information about health topics is increasing, with one in three U.S. adults using the internet to self-diagnose or learn about a health concern . The groThe study purpose was to qualitatively explore the complex relationship between provider HPV vaccination recommendation, media, and vaccine hesitancy among a group of educated mothers who had vaccinated a child against HPV but remained hesitant. Mothers’ perspectives were also elicited on ways to improve the provider/patient HPV recommendation experience in light of the current media environment.The approach used in this study was based on the social-ecological model and the health belief model (HBM). Social-ecological systems theory is a model that underscores how characteristics of the environment influence individual health behavior and outcomes, while the HBM is a social psychological health behavior change model developed to explain and predict health-related behaviors, particularly in regard to the uptake of health services such as vaccination , 24. A pThirty mothers from the Midwest U.S. were recruited for this study. Only mothers were included because research shows that they are most often the primary vaccination decision-makers for their children or share this role with a parenting partner . MothersA semi-structured questionnaire interview guide was developed specifically for this study. It included open-ended questions to elicit discussion about mothers’ decisions about HPV vaccination and was developed by the research team, which was comprised of researchers in the fields of adolescent health psychology, health communication and epidemiology. The original guide was developed to explore the possibility of any social-ecological level influence on mothers’ HPV vaccine hesitancy. The interview began with general questions about mothers’ experiences with the HPV vaccine. Mothers were then asked to recall what they remembered of their experiences and challenges when considering the provider’s recommendation for HPV vaccination and to relay how those experiences shaped what they believe could improve the patient/provider experience with the recommendation. As media influence was brought up by all participants, probing questions addressing the impact of the media were included as the focus of the present study. Specifically, the goals of the current study were to 1) examine media influence as a determinant of decision-making at the time of a provider recommendation and 2) describe mothers’ recommended strategies to cue improved provider recommendations for HPV vaccination within the context of media influences on health seeking and beliefs. The guide was subsequently pilot tested with mothers during preliminary interviews and revised in an iterative manner. Interviews were conducted over the telephone from September, 2019, to February, 2020, and lasted approximately 30 min each. Participants received a $25 gift card to compensate them for the time and effort required for their participation.Interviews were audio-recorded and transcribed verbatim, and thematic content analysis using an established qualitative approach was applied to identify themes and patterns within the data , 27. Muln = 37) of children were females and 53% (n = 42) males.Thirty mothers were recruited  provider gave us the background, but I hadn’t had time to think about it” (Participant 1). Upon initial provider recommendation, all mothers said they had at least heard of the HPV vaccine but recalled negative messages in the media that they had read, seen or heard that contributed to hesitancy or factored into their decision to delay. Online internet exploration via search engines and posts from friends and family on online social media, primarily Facebook, were the primary sources of antecedent exposure. As example: “There wasn’t a lot of research done then (about 4 years ago). He (provider) recommended it that year, and I just wasn’t ready for it yet. Of course, I had gone on the internet and read there were some side effects” (Participant 2). Another mother told of hesitancy from online “stories” on social media: “We waited until 12 or 13. Our kids are really active; I read stories that may or may not be true” (Participant 3). Traditional media  were mentioned less than online sources but were still recalled at the point of provider recommendation by a few. As one mother explained: “I did have reservations; HPV (vaccine) hasn’t been out that long. You hear the pros and cons. … you always hear the worst case scenario on the news” (Participant 4).Mothers reported having their children vaccinated by their family pediatrician at what was typically a well-child visit. All but 5 reported hesitancy upon provider recommendation, with 14 specifically mentioning a decision to delay vaccination. For instance: “Mothers who recalled media messages prior to provider HPV vaccine recommendation revealed three primary themes of hesitancy that stemmed from prior media exposure: safety/side effects, protection/efficacy, and sexual stigma.… there were parents I was reading and hearing about, saying that their kids had a reaction to the shot. That was alarming to me” (Participant 2). The harms mothers spoke of reading about on social media and the internet were varied, ranging from paralysis to autism to general, vague claims of fevers, aches and pains. Most mothers stated they recognized that these claims of harm had no scientific basis, yet some still talked about postings from friends on Facebook that told of a child who became paralyzed or who immediately ran a fever and became sick following vaccination. These stories led some mothers to question whether the HPV vaccine may be harmful, especially if a child already has a health condition. Other than paralysis, most mothers dismissed extreme claims from anti-vaccine groups online.Mothers’ narratives of hesitancy stemming from media messages about the HPV vaccine were foremost related to misinformation about adverse side effects from HPV vaccination, which they described as being widely communicated , especially on the internet and on social media (e.g. Facebook). As one mother said: “There are so many advertisements you don’t know which ones are beneficial. How long has it been around? What are the long-term effects? What it does? What is it supposed to do? Are there negative contraindications?” (Participant 5). Another mother stated, “What are long-term effects of it? There is too much unknown. Knowledge is power for this. If I knew more about this—not from a pharmaceutical company, then I would be more apt (to get it for child)”(Participant 6). One mother said that commercial advertisements were fear-inducing, making her uneasy about vaccine safety:“The advertisement to me (that I remember) is the one with the kid away from school, has fever and can’t make go away. Whether they (media) are making attention, fear is out there. How safe it is was my main concern. It was definitely new with the oldest” (Participant 7).Some mothers also questioned advertisements and commercials on television advocating HPV vaccination because they originated from a pharmaceutical company and “big business”. As a result, these mothers remained quite concerned about adverse effects. For example, “It is media promotion. I think I would have been more secure without that promotion. The commercials – they flooded the networks. Honestly, I question it because the commercials are backed by drug companies” (Participant 6). This mother differentiated that commercials are not the same as traditional news media and should not be trusted. Although commercials were the media most often mentioned in regard to distrust, mothers’ attention to negative media claims about the lack of data supporting long-term efficacy of a “relatively new vaccine”, which were gathered from multiple channels, including traditional and social media, had a couple of mothers wondering whether the HPV vaccine protects from any disease. A few mothers had either only read on the internet or on Facebook that the vaccine only protected from a sexually transmitted infection (STI) and were not aware of its cancer protective benefits, especially for males, at the time of recommendation. For instance, I felt it (HPV vaccine)...from what I had read and knew at the time, was more of a protection for others. I feel it is not as much for him as his future spouse or other”(Participant 8).As with safety, advertisements and commercials on television were also a source of hesitancy regarding the protective benefits of the vaccine. A couple of mothers spoke of distrust of the pharmaceutical-funded commercials, which resulted in questioning the efficacy of and need for the vaccine. For instance, one mother noted, ““From what I have read, I think it (the vaccine) is (equated with being) sexually active. I knew my child was not” (Participant 9). Mothers commonly recalled reading and following conversations from family and friends on Facebook that included strong opinion that allowing one’s child to be vaccinated for HPV would be equivalent to accepting and even encouraging the child to be sexually active. As one mother described of her hesitancy derived from online discussions: “Even if you are leaning about wanting to do the right thing (vaccinate), the  stigma is there  (Participant 10). Also,“There’s so much you read online. I was probably one of those initial ones who quickly attached it to sexual. And thought that (sex) wasn’t going to come now” (Participant 3). Exposure to these online conversations about sexual outcomes was a source of hesitancy for some mothers because they did not want to attach stigma to their child or to themselves as a “bad” mother for agreeing to HPV vaccination. One mother described it this way: “I just didn’t want this to be like my support of him having to jump out and have sex. I was concerned that is this giving him the green light to say yeah” (Participant 11). A couple of these mothers indicated that online conversations about sexual promiscuity had them questioning why the vaccine was “suddenly” needed now (e.g. they wondered if there was a rise in STIs that would prompt need for the vaccine.)Mothers’ stories of their experience with provider HPV recommendation also uncovered that exposure to the internet and social media, particularly Facebook, led to concern and fear of sexual stigma related to the vaccine. Some mothers, as a result of what they had seen discussed on Facebook and online discussions, feared that the HPV vaccine was only for young people who were sexually active. As one mother concluded of the lack of necessity of the vaccine for her child, primarily from her online, internet readings:You read about many more harms that can happen (by vaccinating) than good. Who doesn’t want to help protect their children? It’s more about fear” (Participant 12). Mothers of sons and daughters were equally likely to delay, with a couple of mothers stating they had concerns with injecting a “foreign substance” without confidence of its protective impact. A couple of these mothers of sons delayed vaccination to discuss with their sons and allowed their sons to decide on vaccination.Mothers weighed the uncertainty of the vaccine’s protective effect, safety and sexual stigma gathered from traditional and online/social media sources against provider recommendation and were concerned about the risk of accepting vaccination. One mother described the struggle this way: ““I took some time to think about it (HPV vaccination recommendation); I read up on it on my own. I always feel there is some information they don’t have insight to on commercials. I wanted the medical background. I had to come back for another appointment to have them vaccinated” (Participant 13). Also,“Before vaccinating my 14-year-old, I asked providers I worked with as friends. Providers said they absolutely would vaccinate. I also read information online” (Participant 14).Many mothers who delayed wanted to continue research about the vaccine on the internet. Most looked to sources of information such as WebMD and cancer organizations, but others referred more vaguely to online information seeking from unknown sources. Mothers also mentioned continuation of contact and discussions about HPV vaccination with friends on social media, especially Facebook. These mothers both described how they were exposed to the media prior to provider recommendations and also chose to actively use online information sources after recommendations to address anxiety: “I still have reservations about my (younger) daughter. I need to do more follow up online, to look at the stats--- what is the data showing now? Does it make a difference for kids getting the shots? I want the data. My daughter goes to the doctor today. I am going to be pressured again” (Participant 2).A few mothers who had vaccinated an older child still needed reassurance by seeking information online themselves before vaccinating a younger child. For instance: Mothers indicated that what they had heard, read or seen from advertisements/commercials on television, the internet and social media negatively affected their acceptance of their provider’s HPV recommendation. Hesitation often occurred even when mothers stated they had good relationships with and trusted their provider. Based upon their own experiences with hesitancy from negative media messages, mothers most commonly believed providers could do more to counteract negative media messages so that other mothers might be more likely to accept the HPV vaccine recommendation. Less frequently, mothers also recommended the continuation of provider strategies that helped them to overcome their hesitancies and accept vaccination. The strategies mothers recommended pertained to the timing, delivery and content of the HPV vaccine recommendation in the three areas of safety, efficacy/protection and sexual stigma and are discussed next.“I only did it (accepted vaccination) because I know and trust my physician so much and he could counter the negative messages about side effects I had seen and read” (Participant 12).First, to counteract inaccurate media messages concerning safety, mothers recommended that providers emphasize the statistics surrounding the safety of the vaccine, including how many people have been vaccinated, the year it became available, the studies that support minimal side effects, and the type of testing the vaccine underwent before recommendation. Mothers suggested that providers be prepared with verbal and written messages, primarily in the form of brochures, to counteract the inaccurate, negative messages about adverse side effects reported in the media, especially those related to autism, paralysis and general, vague side effects that proliferate online. One mother described how her physician’s ability to counteract these negative messages influenced her acceptance and recommended that the strategy continue:So, the HPV vaccine, when came up, I did have questions. I had seen on 20/20, Dateline about adverse effects. I was a little more skeptical of HPV than honestly any others because of medical. My question to my pediatrician was,“Did you give to your children?” When she said, yes, I said I am willing to do with my children” (Participant 1).To address efficacy and protection concerns from commercials and social media, mothers suggested physicians communicate HPV vaccine efficacy and need by telling how many people acquire HPV, the types of cancers the vaccine protects, reasoning behind the perceived rise in need for the vaccine, and studies of long-term efficacy. Some mothers recommended that providers emphasize that the vaccine provides long-term cancer protective benefits, given online questioning and distrust of HPV vaccine commercials produced by a pharmaceutical company. Mothers also suggested providers use language that emphasizes cancer prevention over the fear messaging that non-vaccination could leave the child vulnerable to cancer, due to their belief that “everything seen on TV seems to cause cancer.” On the other hand, some mothers described how their provider’s communication about supporting vaccination for their own family members helped them overcome their anxieties. As a result, they suggested providers communicate their own experiences with HPV vaccination  to validate and counteract distrust and skepticism from commercials and online reports. For example, “To combat the stigma associated with the vaccine from misinformation on social media, mothers suggested that providers present the HPV vaccine in one of two framing contexts, either: 1) language that does not address the sexual transmission of HPV or the protection from an STI at all, but only as a protection from cancer or 2) directly, but sensitively, communicating HPV as a sexually transmitted virus alongside an approach that assures your child is not at “fault” but is being protected from a future partner, for which the child has no control over his/her sexual history. Both approaches aimed to de-emphasize or deflect online communication that associates sexual activity with the child.Often I don’t get info until day of shots. It may not be an easy decision unless they can get that information beforehand. They should have a list of pre-questions to ask and be prepared with questions to come in. They need information more than five minutes before they have to have it” (Participant 15). The most common time frame suggested was 1 year in advance; others recommended discussion at age 8 or 9.Because many mothers felt they needed more time to consider the vaccine, they suggested providers should introduce the topic of HPV vaccination as anticipatory guidance, prior to the intended date for which the adolescent is targeted to receive the vaccine. For example: “Parental hesitancy is considered one of the strongest reasons for low HPV vaccination coverage among U.S. adolescents, resulting in missed opportunities for cancer prevention . Indeed,Voices of mothers who are HPV vaccine-accepting but hesitant were uniquely analyzed in this study to show 1) how hesitancy can exist even amid acceptance, and 2) to highlight how exposure to commercial advertising, online media and social media use prior to the provider visit interfere with important HPV vaccination decisions about topics of safety, efficacy and sexual stigma. Online information about vaccines is ubiquitous, and research shows that when individuals encounter a large amount of health information online, it can lead to doubt and unnecessary anxiety and fear –34, whicAs a result of the hesitancy mothers felt from misinformation in the media, mothers suggested strategies for HPV vaccine recommendation communication that may help providers deliver recommendations to facilitate confident acceptance. However, many of the recommended strategies for improving the context and delivery of provider HPV vaccine recommendation, specifically those related to the timing of delivery and participatory approach, are somewhat at odds with the presumptive recommendation approach, which is the consensus preferred communication strategy . While aFurthermore, vaccine hesitancy should not necessarily be viewed as an unchangeable belief. For example, Kornides and colleagues found that secondary acceptance of HPV vaccination is common, with more than two-thirds of parents in a national online survey accepting or intending to accept HPV vaccination after original refusal . They reConfidence boosting and hesitancy reduction strategies may be needed to encourage hesitant, but vaccinating parents to complete the HPV vaccine series and to vaccinate younger children. Unaddressed concerns, particularly those that arise from negative media messages, may lead to increases in hesitancy over time. Additionally, vaccinating mothers who are not confident in their decision are unlikely to serve as strong advocates for HPV vaccination to other parents. In order to counter the false HPV vaccine narratives in the media, providers may need to ensure that they provide mothers with credible, timely information sources, thereby helping parents feel confident in their decision and, perhaps, more willing to vocally support HPV vaccination in their social network.While the qualitative study design allowed for in-depth examination of the topic, there are limitations. Although a sample size of 30 interviews was adequate for this qualitative study, it limits the generalizability of findings, which may not apply to different groups of mothers and geographical locations. The mothers included in this study were highly educated and wanted more information about the HPV vaccine, which may not be true of other groups of mothers. Further, fathers were not interviewed and may have different media consumption patterns that reflect differently on the HPV vaccination decisional process and should thus be studied for comparison. Another future research direction would be to do a comparative study regarding media consumption of mothers who reject HPV vaccination and those who accept vaccination. Additionally, while qualitative research allowed for exploration of attitudes and behaviors surrounding the subject, media causation of hesitancy cannot be claimed. Quantitative approaches are recommended to examine the predictive nature of media information on HPV vaccination hesitancy.Results indicate that educated mothers who accept HPV vaccination for their children may do so with hesitancy due to misperceptions stemming from online media, social media and commercials. Given that media information about the HPV vaccine can be inaccurate and anxiety-producing, providers need to understand how media consumption may influence the way mothers experience and respond to an HPV vaccine recommendation. Providers should be prepared to address maternal fears and misunderstandings regarding safety, protection and sexual stigma. A provider’s recommendation style and technique must also include preparation to counteract negative media messages in order to reduce HPV vaccination delays and hesitancy.Additional file 1:Appendix A. Interview guide.


Human papillomaviruses are the causative agents of cervical and other anogenital cancers along with approximately 60% of oropharyngeal cancers. These small double-stranded DNA viruses infect stratified epithelia and link their productive life cycles to differentiation. HPV proteins target cellular factors, such as those involved in DNA damage repair, as well as epigenetic control of host and viral transcription to regulate the productive life cycle. HPVs constitutively activate the ATM and ATR DNA repair pathways and preferentially recruit these proteins to viral genomes to facilitate productive viral replication. In addition, the sirtuin deacetylases along with histone acetyltransferases, including Tip60, are targeted in HPV infections to regulate viral transcription and replication. These pathways provide potential targets for drug therapy to treat HPV-induced disease. Human papillomaviruses are small, double-stranded DNA viruses approximately 8 kb in length that are the etiological agents of cervical cancers, as well as many other malignancies of the anogenital tract. Over 400 human papillomaviruses (HPV) types have been identified, of which about 200 have been approved by the International Committee on Taxonomy of Viruses (ICTV) to date. Only a small subset of these is associated with human cancers . A groupUp to 1/3 of all HPV types certified by the ICTV infect the genital tract and are sexually transmitted. Approximately 2/3 of young adults acquire HPV infections in the genital tract in the first few years of sexual activity ,6. Most HPVs infect cells in the basal layer of stratified epithelia and establish their genomes as low-copy nuclear episomes. Productive replication and assembly of progeny virions only occurs upon differentiation. HPV genomes can be subdivided into three functional regions: the upstream regulatory region (URR), the early (E) region, and the late (L) region .The URR region does not code for any proteins and encompasses almost an eighth of the entire HPV genome. It contains the viral origin of replication (ORI) and the early promoter regulatory elements. The other two regions encode approximately eight open reading frames (ORF) that are expressed from polycistronic mRNAs. The E region encodes for the HPV early genes E1, E2, E4, E5, E6, and E7. These early genes have critical functions in the viral life cycle, including HPV genome replication, gene expression, immune system evasion, and viral genome persistence . The twop110, p107 and p130 (pRb2), with functions in cell cycle control and transcription .,65.33,65The E7 protein acts in part through activation of the innate immune transcriptional regulator, STAT-5, which increases expression of TOPBP1, an ATR activator, and also activates ATM . ActivatThe cohesin protein SMC-1 can also be phosphorylated by ATM and plays a critical role in the homologous recombination repair process that requires a sister chromatid template. SMC1 is constitutively phosphorylated in both undifferentiated, as well as differentiated HPV-positive cells ,69, and The Fanconi anemia (FA) pathway also provides critical functions for DNA repair. The FA pathway repairs interstrand cross-linked DNAs and is regulated by both the ATM and ATR pathways. ATR phosphorylates several proteins in FA pathway and this is required for optimal FANCD2 monoubiquitination and activation. ATM can also phosphorylate FANCD2, but this leads to an S-phase arrest . FA patiFA proteins are intimately linked with the HPV life cycle and the development of HPV-related neoplastic disease. For example, loss of FANCA and FANCD2 leads to the accumulation of the high-risk E7 proteins in keratinocytes . This inActivation of ATM in response to DNA damage is correlated not only with rapid autophosphorylation, but also its acetylation . The resTip60 levels are increased in HPV-positive keratinocytes that maintain complete viral genomes . In contSirtuins are a family of lysine deacetylases with homology to the budding yeast, silent information regulator 2 (Sir2), which regulates the formation of heterochromatin through the deacetylation of lysine residues on histone tails . The humSIRT1 is the most extensively studied member of the sirtuin family and deacetylates histones H3, H4, and H1 . It has SIRT6 also has a multitude of reported functions in DNA damage repair, promoting resistance to DNA damage and suppressing genomic instability. SIRT6-deficient cells display marked genomic instability and are hypersensitive to DNA-damaging agents, such as ionizing radiation, methylmethane sulfonate, and hydrogen peroxide . Like otSIRT2 is the only Sirtuin found in the cytoplasm during most of the cell cycle, but translocates into the nucleus during mitosis . SIRT2 eSIRT1 is the only Sirtuin that has been studied in relation to HPV biology. SIRT1 levels are increased in HPV-positive cells through the combined action of the E6 and E7 oncoproteins . Knock-dHPV proteins activate a number of cellular pathways to facilitate viral replication. This includes those involved in DNA damage repair, as well as epigenetic control of host and viral transcription. HPVs constitutively activate the ATM and ATR DNA repair pathways and preferentially recruit these proteins to viral genomes. Both these pathways are required for productive viral replication. Epigenetic modifiers that regulate acetylation also play important roles. The sirtuin deacetylases, along with histone acetyltransferases including Tip60 are required in HPV infections to regulate viral transcription and replication. These pathways are also important as they could be useful targets for drug therapy to treat HPV-induced disease.


Plants are an important source of chemically diverse natural products that target microtubules, one of the most successful targets in cancer therapy. Colchicine, paclitaxel, and vinca alkaloids are the earliest plant-derived microtubule-targeting agents (MTAs), and paclitaxel and vinca alkaloids are currently important drugs used in the treatment of cancer. Several additional plant-derived compounds that act on microtubules with improved anticancer activity are at varying stages of development. Here, we move beyond the well-discussed paclitaxel and vinca alkaloids to present other promising plant-derived MTAs with potential for development as anticancer agents. Various biological and biochemical aspects are discussed. We hope that the review will provide guidance for further exploration and identification of more effective, novel MTAs derived from plant sources. Microtubules are the major components of the eukaryotic cytoskeleton. They are composed of α- and β-tubulin heterodimers that interconvert between phases of rapid growth (polymerization) and shrinkage (depolymerization) . MicrotuThe crucial roles played by microtubules in both mitotic and interphase cellular functions make them important anticancer drug targets. Accordingly, microtubule-targeting agents (MTAs) inhibit the proliferation of cancer cells by disrupting interphase cell signaling events and/or preventing the precise functioning of spindle microtubules, both of which ultimately induce cell death via apoptosis . Notably®) and vinca alkaloids, which are important treatments for many different cancers, such as ovarian, breast, bladder, prostate, and lung cancers, and lymphoma [The best examples of plant-derived MTAs are paclitaxel  had been obtained from various Tacca sp. or through semi-synthesis [Tacca plants -(R)-persin) is a polyketide long-chain lipid with strong structural homology to linoleic acid, and is synthesized in idioblast oil cells present in avocado leaves and fruit . Persin Persea americana Mill. (Lauraceae) [2/M arrest, and also synergized with other microtubule stabilizing agents in ovarian cancer cells [Although persin was first isolated in 1975 from the leaves of the avocado plant, uraceae) , its micuraceae) . In the uraceae) ,36,37,54er cells . Other Mer cells ). Thus, The exact binding site of persin on tubulin is not known. However, a cell-based Flutax-1 (a fluorescent taxol derivative) competitive binding assay showed that persin displaces Flutax-1 from tubulin, suggesting that persin may occupy a site adjacent to or overlapping with the taxoid-site on β-tubulin . Althoug1 and 10, reduction in the length of side chain is the only structural difference (1 exhibited significant antiproliferative effects (with an IC50 of ~17 μM); whilst 10 was devoid of any antiproliferative effects even at concentration as high as 65 μM [10, these compounds bind differently to its tubulin target, which, in turn, might be contributing to their better bioactivity vs. that of 10.Several analogues of the persin have been synthesized A,B, and fference A,B. In bas 65 μM  , ferrocenyl methylene curcuminoids , and the ferrocenyl ethanone curcuminoids (compounds 14 and 15), showed much better tubulin polymerization inhibition acitivity than curcumin , (2) an isoxazole derivative of curcumin (compound 20), and (3) a benzylidiene derivative of curcumin (compound 21) [16–19) and isoxazole (compound 20) curcumins docked in to the curcumin binding site on tubulin and showed a greater increase in stability than curcumin at physiological pH and reducing atmosphere [21) bound tubulin with a higher affinity compared to curcumin, and it was associated with increased ability to prevent microtubule assembly and induce cancer cell death. The 21 has free diketone moieties and a substituted polyphenol ring in between the dicarbonyl moiety, suggesting that the extra steric hindrance caused by the substitution of a polyphenol in between the diketones may make the compound conformationally more favorable to bind with tubulin. Alternatively, substitution of a polyphenol ring in between the diketones may introduce a tridentate molecule that can bind tubulin with higher affinity.Chakraborti et al. also synthesized several curcumin analogues  Figure . They inound 21)   is one of most active curcumin derivatives [22 inhibited the microtubule assembly, perturbed the lattice structure of microtubules, suppressed their GTPase activity, and inhibited cancer cell proliferation much more effectively than curcumin [22 also showed more stability in aqueous buffer than curcumin [22 may be partly due to its increased stability in solution.Curcumin-derived compound C1  . StructuPapaver somniferum [Noscapine is a non-narcotic natural phthalideisoquinoline alkaloid that is originally isolated from the opium poppy mniferum   or clinical response .Moreover, most noscapinoids are not substrates for the Pgp drug efflux pump and several of them showed synergistic interaction with other MTAs ,83. NoscNoscapine and its derivatives bind tubulin at or near the colchicine site, and inhibit microtubule dynamics without causing gross depolymerization of microtubules ,84.A number of noscapinoids with antiproliferative effects superior to the parental noscapine have been synthesized recently . One notMaytenus Serrata and later from the bark of Maytenus buchananii [Maytansinoids are MTAs derived from maytansine C. Maytanchananii ,85  on β-tubulin that is different from the vinca domain . The mayThrough a semi-synthesis strategy, a number of maytansine analogs  that have disulfide or thiol groups that favor covalent linkage with monoclonal antibodies have been obtained ,89,90. MAdditional plant-derived microtubule destabilizing agents that exhibited bioactivity in in vivo tumor xenograft models or that have undergone clinical trials include chalcones and quercetin ,92. ChalThey occupy the colchicine site on tubulin and prevent microtubule polymerization ,91. BesiMicrotubule cytoskeleton is one of the most succesful targets in cancer therapy, and MTAs, especially paclitaxel and vinca alkaloids, play a dominant role in the treatment of diverse cancer types. However, the development of resistance by cancer cells, the reduced pharmacokinetic profile, and the severe side effects associated with their formulations often present challenges to the clinical applicability of the MTAs. Several techniques have been recently developed to avoid these issues, including nanoparticle delivery, covalent linkage to the fatty acid docosahexanoic acid, encapsulation in lipid complexes, and conjugation to antibodies. While these techniques are possibly a good approach for older generation MTAs, the future of MTAs probably lies with newer agents that more effectively evade these serious problems.Plants are a major source of MTAs. The search for novel MTAs from plants is necessary, as it will provide a greater range of lead compounds. Notably, the production of biologically active secondary metabolites in plants are less affected by both abiotic  and biotic  factors, compared to marine organisms and microorganisms . MoreoveTo date, only a small portion of the world’s plant biodiversity has been exploited for the search for MTAs, thus indicating that there exists a huge opportunity for the discovery of novel MTAs from plants. Although major challenges, such as access and supply, complications in high-throughput screening due to the complexities of plant extracts, and the high cost of creating plant collections limit the interest in the search for MTAs from plants, recent advances in synthetic methodologies, fractionation and analytic methods, and genetic engineering provide hope to effectively overcome some of these challenges, including the supply issues and the screening difficulties from crude and pre-fractionated extracts. It seems that the future of plant-derived MTAs likely depend on the key features that include robust anticancer activity, low toxicity to normal tissues, and the ability to overcome resistance to existing MTAs. Identification of MTAs with these ideal features from plants would undoubtedly benefit the development of potential MTAs for cancer therapy.As described herein, the plant-derived MTAs, taccalonolides, persin, curcumin, combretastatins, noscapine, maytansine, chalcones, and quercetin, have exhibited improved anticancer properties when compared with the taxanes and vinca alkaloids. Of note, curcumin, combretastatins, noscapine, maytansine, and quercetin have already undergone clinical trials evaluating their efficacy against various cancers ,63,87,93


Common blue nevi tend to be singular or localized, with multiple eruptive blue nevi being a rare occurrence. We report the case of a young adult who presented with multiple asymptomatic lesions that had appeared gradually over a few years. Physical examination revealed 30 distinct, blue-gray macules diffusely over the medial buttocks, lower back, and dorsal arms. Histopathology showed pigmented dendritic melanocytes with associated melanophages, features characteristic of blue nevus. This case demonstrates that eruptive blue nevi can present as numerous, disseminated lesions over multiple anatomic sites. Recognition of the various patterns of eruptive blue nevi and their benign nature can reduce unnecessary biopsies and work-up. Common blue nevi occur in about 0.5-4.0% of healthy white adults, while multiple eruptive blue nevi are rarer and have been described in only 13 case reports so far . These cA young adult presented to dermatology with multiple, discrete round small blue-gray macules that had first appeared a few years ago. The patient was otherwise healthy except for a history of scoliosis. The macules had initially appeared on the bilateral buttocks and then on the lower back and arms. The lesions were asymptomatic and there was no reported trauma to the area nor any tanning-bed use. There was no family history of similar lesions. On exam, 30 distinct, blue-gray, and well-demarcated macules, ranging in size from 1 mm to 3 mm, were found on the medial buttocks Figure .Diagnosis of blue nevi can be made clinically, although atypical and challenging cases may require a biopsy to confirm the diagnosis. Histologically, the blue nevi are located in the mid-dermis and are thought to arise from dendritic melanocytes that have not migrated to the epidermis from their site of origin in neural crest cells -4. They Types of blue nevi, based on clinical and histopathological differences, include common and cellular -3. CommoAmong the 13 cases of eruptive blue nevi reported so far, only two had multiple areas involved, namely a patient with bilateral tibial involvement and a second with diffuse distribution over the body ,6. HowevThe cases of eruptive blue nevi were associated with a known or possible trigger, although there may have been a selection bias for reporting such cases. Triggers or associations have included trauma, sunburns, oral contraceptives, and vesiculobullous dermatoses -3. In adIt is important to distinguish blue nevi from satellite metastatic melanoma or regressive melanoma . Common Given limited data in the literature, our case report provides an example of eruptive blue nevi that can be benign even when numerous, disseminated over multiple anatomic sites, and sudden in appearance. The recognition of unique, benign patterns can reduce unnecessary biopsies and provide reassurance to patients. Further long-term follow-up studies are needed to truly assess the risk of malignant transformation.