Mutational and functional analyses of the polyketide-tailoring cytochrome P450 monooxygenases, and PKS phylogenies give contradictory clues on potential evolutionary scenarios . The KS-AT linker proves to be well suited as fusion site for both excision and insertion of modules, which supports a model for alternative module boundaries in some PKS systems. This study teaches important lessons on the evolution of PKSs, which may guide future engineering approaches.Polyketides produced by modular type I polyketide synthases (PKSs) play eminent roles in the development of medicines. Yet, the production of structural analogs by genetic engineering poses a major challenge. We report an evolution-guided morphing of modular PKSs inspired by recombination processes that lead to structural diversity in nature. By deletion and insertion of PKS modules we interconvert the assembly lines for related antibiotic and antifungal agents, aureothin  for aurothin and neoaurothin biosynthesis, and reveal engineering site suitable for diversifying PKS systems. Whereas their chemical synthesis is often challenging, bacteria produce these valuable compounds from simple carboxylic acid building blocks by means of modular (type I) polyketide synthases (PKSs)2. Each module of these highly versatile assembly lines consists of a minimal set of domains for chain elongation: a ketosynthase (KS) catalyzing Claisen condensations of the activated acyl and malonyl building blocks, an acyltransferase (AT) for selecting and loading extender units, and an acyl carrier protein (ACP) domain that serves as an anchor for the growing chain3. Additional β-keto processing domains for ketoreduction (KR), dehydration (DH), and enoyl reduction (ER) increase the structural diversity of the polyketide chain. The architecture of the megasynthase is typically arranged in a way that each module catalyzes a single chain elongation, ensuring that polyketide assembly progresses in a unidirectional manner until the full-length product is released by a thioesterase (TE) domain3. Consequently, in most bacterial type I PKSs the PKS assembly line directly corresponds to the chemical structure of the resulting polyketide.The wealth of complex polyketides produced by bacteria is an essential source for antifungal, antitumor, antiparasitic, and immunosuppressive agents that are currently used in the clinics4. Likewise, the assembly line logic inspires the rational reprogramming of PKSs2. In many cases, such engineering attempts lead to the production of natural product derivatives6 and provide insight into the mechanisms of polyketide assembly7. However, routine PKS engineering is challenging not only because of constraints in DNA manipulation and recombineering8 but also because of the intricacies and complex dynamics of the megasynthases. For a holistic view on the molecular machineries insight into catalytic dynamics, spatial constraints, potential incompatibilities, and substrate specificities of the catalytic domains is required. It is not surprising that many combinatorial biosynthesis approaches in a mix-and-match fashion are unsatisfactory, as domains were swapped or recombined neglecting the structural impact, including non-functional regions, on protein–protein interactions. Consequently, domains and/or modules of an engineered PKS are impaired or simply non-functional9, which results in dramatically reduced yields or complete loss of production10.The paradigm of successive assembly and co-linearity between assembly steps and final product sets the basis for the in silico prediction of polyketide structures from type I PKS genes, thus enabling the discovery of natural products by genome mining11. Extensive bioinformatics analyses suggest that nature harnesses point mutations, gene duplication, gene loss, homologous recombination, and horizontal gene transfer in the evolution of type I PKSs15. However, there is little experimental evidence for such mechanisms being at work in the evolution of modular PKSs. Emulating evolutionary processes could address some of the key questions in PKS research: What are the prerequisites for the morphing of one pathway into another? Which impacts have polyketide-tailoring enzymes that coevolved with the scaffold-assembly lines? Which sites are suited for recombinations and fusions and allow the reprogramming of polyketide assembly lines?Regardless of current constraints in PKS engineering, nature provides us with successful strategies to diversify polyketide structures through evolutionary processes1)16 and neoaureothin (2)17 are used as model in a proof-of-concept study. Both the aur PKS and nor PKS  as starter unit18, and in both pathways homologous PKS modules generate polyene-pyrone backbones20, which are then subjected to further enzymatic tailoring by O-methyltransferases (AurI/NorI) and cytochrome P450 monooxygenases (AurH/NorH)21. A remarkable feature of the aur and nor PKSs is that both assembly lines contain iteratively acting domains and modules24. The only difference between the two pathways is the size of the products’ polyene backbones; compared to 1, compound 2 is composed of two additional propionate units, which are introduced by means of two additional modules in the nor PKS  in the NCBI  database identified several gene loci with high homology to the aur and nor gene clusters  deviates from the others as it lacks a norB/aurB homolog. The absence of this gene and the correct assembly of the contigs were confirmed by four pairs of PCR primers 28. This low yield might result from the non-concerted expression of the nor biosynthesis genes using a three-plasmid system. To increase neoaureothin production we optimized the heterologous expression system to reassemble the nor gene cluster in a continuous gene region  was integrated into the genome of the heterologous expression host S. albus by site-specific recombination. The complete nor gene cluster was then obtained by a homologous recombination using a suicide vector (pYU93) harboring the left part of the gene cluster. The resulting strain  produced threefold higher titers (45 mg L−1) of neoaureothin compared to the previous construct , (AurB/NorB), and (AurC/NorC) share high identities on the DNA and amino acid levels, we excised the gene regions for modules 2 and 3 in the nor gene cluster. Thereby, it was essential to consider the compatibility of the docking domains between the individual PKS proteins33, because the sequence elements at the extreme C- and N-termini of PKS subunits help mediate their interactions. The amino acid sequence alignment among PKS proteins indicated that the CDD  of AurA/NorA and the NDD  of AurB/NorA′ is a class 1a docking domain system  lacking norA′  and trace amounts of 7-hydroxydeoxyaureothin (3), but not aureothin (1) , the second one has a fusion site at the KS-AT linker region (aur* PKS)  Fig. . ApparenrA′ Fig.  and tracrA′ Fig.  and trac5, Supplementary Figs. 3) but no aureothin (1) could be detected  compared to AurA alone24, indicating that the docking domains promote protein interactions. However, the low production of the full-length polyketide pointed to incorrect protein folding, likely because of suboptimal fusion sites. Thus, we revisited the fusion site in the aur* PKS and found that the recombinant KS-AT linker in the aur* PKS bore four amino acids more than that in the genuine aur PKS  could still not be detected by HPLC. Instead, we noted the formation of other metabolites  and 7-deoxyaureothin (4), which differs only from 1 in that they do not form the tetrahydrofuran ring  from the nor pathway. NorH is only able to convert deoxyaureothin (4) into the hydroxylated congener 3, whereas the second oxidation and thus also heterocyclization do not take place  needed to adjust.From in vivo and in vitro studies we know that the formation of the tetrahydrofuran ring is the last step in aureothin biosynthesis and that its installation involves two sequential C-O-bond formations catalyzed by a single cytochrome P450 monooxygenase, AurHace Fig. . In case39. Threading of the NorH amino acid sequence onto the AurH crystal structure revealed a conserved hydrophobic pocket for binding the pyrone ring of deoxyaureothin. However, the modeling indicated that NorH has a wider cavity around the active center, as some amino acid residues possess smaller or more flexible side chains than those found in AurH , and recognize similar substrates. Thus, the overall structure of NorH is likely similar to AurHurH Fig. . Based onorH variants, including the I19F, V71L, T291L, T292P, W317F, and T392L mutants, and cloned them individually into expression vectors for complementation of an aurH knock-out mutant (ΔaurH)37 . In contrast, the mutant complemented with NorH partly transformed 4 into 3, and it was not capable of forming 1 (negative control) All ∆aurH mutant strains complemented with point-mutated NorH variants showed the same chemotype. The only difference was a slightly increased 3-to -4 ratio for NorH-V71L and NorH-T292P   to aureothin (1), albeit only incompletely . Yet, in the metabolic profile of the ∆her Fig. . Thus, wher Fig.  and atteely Fig. .aur-to-nor PKS evolution.Taken together, structure-based, rational mutations and domain swapping of NorH are not sufficient to reconfigure the active site of NorH to function like AurH. The 70% C-terminal AurH hybrid NorH (Abc) shows only substantially reduced activity. These results indicate that complex evolutionary processes would be required to maintain THF ring formation activity when mutating NorH to AurH. Consequently, we also considered the reverse scenario and aimed at emulating a potential 6) into 2. To this end, norH was deleted in S. albus_nor PKS using the λRed system. The resulting ∆norH mutant was fermented, and the culture extract was analyzed by HPLC. The HPLC profile showed that ∆norH lost the ability to produce 2. Instead, a different metabolite was detected , and the other one in the KS-AT linker region (nor* PKS)  Fig. .Fig. 5Ovnor# PKS), we generated a fusion site at the N-terminal docking domain region of aurB and PCR-amplified intact norA′ including its N- and C-terminal docking domains (norA′#) , we chose a fusion site within the KS-AT linker region of AurB, and amplified the gene fragments for the region between the NorA′-AT2 domain and the NorB-KS4 domain (NorA′*-NorB*) for recombination , over-oxidized 11a-hydroxyneoaureothin (7), non-reduced 7-deoxy-7-dehydroneoaureothin (10), non-reduced 2-pyrone-7-deoxy-7-dehydronoeaoreothin (9), and 2-pyrone-4-desmethyl-7-deoxy-7-dehydronoeaoreothin (8). The structures of all compounds were elucidated by NMR analyses  in which the N-terminal docking domain of AurB was swapped to that of NorB without inserting NorA′#  into aureothin (1). The homolog AurH, however, was found to have broader substrate specificity than NorH and readily transforms the deoxy precursor of neoaureothin into the final product.In this study we used the related 57, the substrate specificities of the oxygenases may indicate that AurH is more ancient than the more specialized NorH  PKS morphing by insertion of two modules corroborated the revised model, although it contrasts with the conclusion drawn from the phylogeny. This is a rare example of successfully engineering two additional modules into a PKS and shows that the aur and nor gene cluster share the same evolutionary origin.According to the model of generalist-to-specialist enzyme evolutionorH Fig. . Furtheraur/nor engineering experiments are that (1) evolutionary models guide the morphing of modular PKSs from two distinct biosynthetic pathways into another, and that (2) the specificity of tailoring enzymes may give additional clues on the direction of evolution. Phylogenies alone should be interpreted with care. The third important lesson relates to the design of functional PKS constructs; (3) the KS-AT linker is particularly well suited as engineering site for both excision and insertion of modules. As in a plethora of other, mainly unpublished, studies, engineering the aur and nor PKSs has been a tour de force because of suboptimal recombination sites that led to less active or fully non-functional constructs. In our efforts to alter the aur and nor PKSs we found that best results for excising or inserting modules were obtained when the recombination sites were located in the KS-AT linker region. Only in this way it has been possible to create a nor* PKS that produces the aureothin backbone, and an aur** PKS assembling the neoaureothin scaffold. Still, the product yields of the engineered aur**_PKS (aureothin backbone) and the nor*_PKS (neoaureothin backbone) are substantially lower (10–20-fold) than those of the corresponding native assembly lines, and could be optimized by further adjusting the KS-AT linker region59, for example. It should be pointed out that the constructs with recombination sites within the docking domains (aur# PKS and nor# PKS) were non-functional.The general lessons learned from the 60. Notably, in the alternative model the boundary for each module is the KS-AT linker, which in our case proved to be the best site for adding or deleting modules . Recently, a platform has been established that allows a swift recombination of NRPS modules to create artificial peptides65. Interestingly, for the de novo design and engineering of NRPSs this strategy employs the C-A (condensation-adenylation) linker region as fusion point66, which corresponds to the KS-AT linker in PKSs.Our results of the engineering experiments, specifically the location of the fusion sites, are particularly intriguing in light of a recently proposed revision of the PKS module boundaries. Contrary to the textbook architecture of modules (KS-AT-X-ACP), in the phylogeny-based model a PKS module would start with an AT domain and end with the KS domain that was previously assigned to the downstream module (AT-X-ACP-KS)les Fig. . TherefoIn conclusion, our work is a proof-of-concept for a successful evolution-guided reprogramming of modular PKSs. Beyond insight into nature’s chemical diversification by evolving PKS and post-PKS enzymes, the knowledge gained supports a paradigm for module boundaries for at least some assembly lines, which may advance the field of rational PKS engineering and synthetic biology.E. coli strains TOP10 (Invitrogen) and XL1-Blue (Agilent) were used, E. coli HB101/pRK2013 for triparental conjugation67, and BW25113/pIJ790 for PCR-targeting procedures68. For plasmid selection, E. coli strains were cultured in Luria-Bertani (LB) medium supplemented with spectinomycin , apramycin , kanamycin  or chloramphenicol . S. albus  was used as host strain for heterologous expression experiments. For metabolite production, Streptomyces strains were grown at 28 or 30 °C with 150 or 180 rpm orbital shaking in TSB, R5  or J-medium  for 2 days. Seed cultures (10 mL) were used to inoculate 100 mL of the up-scaled culture in R5, modified R5 (R5 medium without sucrose and 1 g L−1 casamino acids) or M10 medium . The fermentation was stopped after 5 days cultivation . For sporulation, Streptomyces strains were grown on MS (mannitol soya flour) agar plates for 7 days. For conjugation MS agar plates supplemented with MgCl2 (10 mM) were used, which were overlaid after one-day incubaction with nalidixic acid  and appropriate antibiotics.For routine subcloning, E. coli69. Restriction enzyme digested and PCR-amplified DNA fragments were purified from agarose gel using the Monarch DNA Gel Extraction Kit (New England Biolabs).All routine experiments such as PCR, DNA isolation, plasmid preparation, restriction digests, gel electrophoresis, ligation, and transformation were performed according to standardized methods for Streptomyces avermitilis, pte; avermectin, ave) were aligned using the GUIDANCE2 Server26. Alignment columns with a score below 0.93 were removed from the alignment. Tree reconstruction was done by the method of Bayesian inference using version 3.2.6 of the MrBayes software27. The calculation used the model jumping option and was run with four independent chains for 1.5 million generations sampling one tree per 1000 generations. Trees were summarized with a burnin of 300 trees.The amino acid sequences of the KS domains of the aureothin and neoaureothin gene clusters and other selected actinobacterial PKS clusters  (Stratagene) was blunted and ligated into AfeI-digested pYU0128, in which aurD was cloned downstream of constitutive promoter, actII-ORF470, to yield the plasmid pYU91. The 34.5 kb XbaI DNA fragment from pNT8728 was cloned into SpeI-digested pYU91. The resulting suicide vector pYU93 was introduced into S. albus::pNT42 containing norB, C, and I by triparental conjugation to give the recombinant strain S. albus::pNT42/pYU93 .The 0.8 kb norA′ gene, primer pair NorA′-HpaI-fw (5′-CCGGACGTCCTGCCACTGCGCTTCGGCGCCTGAGCGAGCGTTAACGAACACGTAGAAAGCCAGTC-3′) and NorA′-HpaI-rv (5′-GCAGCCCTTCGGCGTCCAGTCGGTCGATGGAGCCGGGTTAACTTGGTCGGTCATTTCGAACC-3′) were used to amplify the kanamycin resistance cassette from pK1971. The obtained amplicon was introduced into E. coli BW25113/pIJ790 harboring pYU93. The targeted norA′ gene was replaced by the kanamycin resistance cassette through the λRED system to generate pYU93 ΔnorA′-Kan. This plasmid was restricted by HpaI and then self-ligated to generate pYU93 ΔnorA′. This plasmid was introduced via triparental mating into S. albus::pNT42 to generate S. albus::pNT42/pYU93 ΔnorA′ .To remove the XbaI DNA fragments from pYU4828 and pYU6928 were individually ligated into SpeI-digested pYU91. The resulting suicide vectors pYU95 and pYU98 were introduced into S. albus::pNT42 to generate S. albus::pNT42/pYU95  and S. albus::pNT42/pYU98 , respectively.The 23.7-kb S. albus_aur** PKS mutant was constructed by a PCR-targeting approach. To adjust the length of the KS-AT linker region in norB**, primer pair KS2_fw_HpaI_2 (5′-GTCATCCTGGAACAGGCCGAGCAGCCCCCGGCCGAGGACGTTAACGGAACTTCGAAGTTCCCGCC-3′) and KS4_rv_HpaI_2 (5′-CAGGGGCAGGCCCACGGCCGGGATGTCCGCGCCCTCCGGGTTAACGGAATAGGAACTTCATGAGC-3′) were designed and used to amplify the spectinomycin resistance cassette aadA from pIJ778. The 1.4-kb PCR product was introduced into E. coli BW25113/pIJ790 harboring pNT87. Mediated by the λRED system, the targeted DNA region was replaced by the aadA cassette to generate pYU71. pYU71 was digested with HpaI to remove the aadA cassette and then self-ligated to yield pYU72. The 23.6 kb XbaI DNA fragment was cut out from pYU72 and cloned into SpeI-digested pYU91 to obtain suicide plasmid pYU99. Finally, pYU99 was introduced into S. albus::pNT42 to generate S. albus::pNT42/pYU99 .The norH subcloning. The QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) was used for site-directed mutagenesis. The E. coli-Streptomyces shuttle vector pMR1738 was used for norH expression in S. albus::pHJ68 (∆aurH)37 and as the template for site-directed mutagenesis. The primers for site-directed mutagenesis are listed in Supplementary Table The Zero Blunt PCR Cloning Kit (Invitrogen) was used for Five pairs of partially overlapping primers (Supplementary Table FspI in the corresponding genes) located at the N-terminal end of I helix  with primers Kan-NorH-fw-1 (5′-GCGGCAGCAGGCCGCACCCTTGAGCGAAGGACCGTGTTACTGGGCTATCTGGACAAG-3′) and Kan-NorH-rv-1 (5′-GCCCTTCAACCGGAGTTGAGATATCCGATGGCTCGCCCTGATGCGGTGTGAAATAC-3′). The PCR product was introduced into E. coli BW25113/pIJ790 harboring pYU93 to generate recombinant and suicide plasmid pHY42. pHY42 was introduced into S. albus::pNT42 to yield norH knock-out mutant S. albus::pNT42/pHY42 (ΔnorH).The PCR-targeting approach was used to replace the gene E. coli‐Streptomyces shuttle plasmid pHJ4819 containing the aur gene cluster was digested with KpnI. The 15 kb DNA fragment was inserted into the pCR-Blunt vector to yield pHY115, which was used for further gene cloning steps. The PCR-targeting approach was used to insert the spectinomycin resistance cassette aadA into the point mutation site. The aadA cassette was amplified from pIJ778 with primer pair Spec-norA′-fw (5′-GGGCCGGACGCCCTGCCGCTGCGCTTCGGCGCGGCCTGAAATATTATTCCGGGGATCCGTCGACC-3′) and Spec-norA′-rv (5′-GGGGTAGCGACATCCCATTGACACCACCGCGACGGGCTCCTTAAGTGTAGGCTGGAGCTGCTTC-3′), which also introduced SspI and AflII restriction sites into the amplicon. The 1.5 kb aadA cassette was introduced into E. coli BW25113/pIJ790 harboring pHY115. Then, this cassette was inserted into pHY115 by homologous recombination, yielding pHY119. The 16 kb KpnI-digested pHY119 fragment was inserted into KpnI-digested pHJ48-oriT fragment to obtain pHY124. The gene region for the N-terminal docking domain region of AurB was replaced by that of NorB in pHY124 to generate pHY129. A DNA fragment norA′# including the gene regions for the N- and C-terminal docking domains was constructed as follows: The main part of norA′ was cut out from pYU93 by EcoRI and EcoRV, and sequences upstream and downstream of the main part of norA′ were PCR-amplified. The three DNA fragments were ligated into the pCR-Blunt vector to obtain pHY125. The 11 kb DNA fragment was cut out from pHY125 by XbaI and EcoRI and ligated to pBU1223/XbaI + EcoRI fragment to yield pHY132. pHY129 and pHY132 were co-introduced into S. albus by triparental conjugation to generate S. albus::pHY129/pHY132 .norA′* fragment harboring the gene region coding for the norA′-AT2 domain to norB-KS4 domain, and the KS-AT linker in aurB was chosen as insertion site. The strategy to induce the HpaI and SpeI restriction site in pHJ48 was the same as above, with primer pair Spec-aurB-fw (5′-CCACCTGATCCTCGAACAGGCCCCCGATGCGGAGGAGTCGGTTAACAT TCCGGGGATCCGTCGACC-3′) and Spec-aurB-rv (5′-GGATGCGTGCGGGCGCGCCGGACGCGGGCTCCGCGTCACTAGTTGTAGGCTGGAGCT GCTTC-3′), and the resulting aadA cassette was inserted into pHJ48-oriT to yield pHY133. A DNA fragment coding for the norA′-AT2 domain to norB-KS4 domain was cloned and inserted into the pCR-Blunt vector to produce pHY121. The 11-kb HpaI/SpeI-digested DNA fragment of pHY121 was ligated into the HpaI/SpeI fragment of pHY133. The resulting plasmid (pHY134) was introduced into S. albus by triparental conjugation to generate S. albus::pHY134 .The S. scabrisporus (DSM41855) was cultured in TSB medium (25 mL) with orbital shaking at 30 °C for 3 days. The genomic DNA was purified by the Wizard® Genomic DNA Purification Kit (Promega). The joint region between norA′ and norC was PCR-amplified using four primer pairs: NorAAfw-Ss (5′-TGCGATGGCGCTGCACGACG-3′)/NorCrv-Ss (5′-GACAACACACTGGCGGCGGC-3′), NorAAfw-Ss/NorAArv2-Ss (5′-CAACGACGCCGAACA CGCCG-3′), NorAAfw2-Ss (5′-GCCTGCTGGACTCGCTGCTG-3′)/NorCrv-Ss, and NorAAfw2-Ss/NorCrv2-Ss. NorAAfw-Ss and NorAAfw2-Ss are derived from norA′, while NorCrv-Ss and NorCrv2-Ss are derived from norC. The PCR reaction was performed by KAPA 2 G fast HotStart DNA polymerase (KAPABIOSYSTEMS) with 2.5% DMSO.Strain 3 or CDCl3/CD3OD were measured on Bruker Avance DRX 500 or 600 MHz spectrometers equipped with a cryo probe at 300 K. The residual solvent peak  was used as reference. LC-HRMS measurements were carried out on a Thermo Fisher Scientific Exactive Orbitrap equipped with an electrospray ion source. Column: Betasil 100-3 C18 (2.1 × 150 mm); elution gradient: solvent A: water + 0.1% formic acid, solvent B: acetonitrile, gradient: 5% B for 1 min, 5–98% B in 15 min, 98% B for 3 min, flow rate: 0.2 mL min−1.NMR spectra of compounds dissolved in CDCl−1). Aureothin (350 nm), 7–deoxyaureothin (350 nm), 7-hydroxydeoxyaureothin (350 nm), neoaureothin (378 nm), intermediate (338 nm), and 7-deoxyneoaureothin (378 nm) productions were quantified by calculating peak area sizes compared to those of references.To the bacterial culture 1 V ethyl acetate was added, and the mixture was stirred at room temperature overnight. Then, the organic phase was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was dissolved in 1 mL methanol. The obtained extract (10 µL) was analyzed by reversed-phase column HPLC  using a gradient program (solvent A (water + 0.1% trifluoroacetic acid) and solvent B (acetonitrile), 20% B for 5 min, to 99% B in 30 min and kept for 5 min at a flow rate of 1 mL minS. scabrisporus DSM41855 was cultured on five PDA (potato dextrose agar) plates for 7 days. Agar was chopped and stirred in 100 mL ethyl acetate overnight. The extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was dissolved in 0.5 mL methanol. The reversed-phase HPLC analysis was performed as described above.S. albus::pNT42/pYU93 ΔnorA′ (aur§_PKS) culture (1.5 L) was filtered through Celite 545. The water phase was further extracted with ethyl acetate (2 × 1.5 L). The combined extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was subjected to silica gel column chromatography  using a dichloromethane/methanol gradient. The dichloromethane-eluted fractions were subjected to preparative RP-HPLC , B (83% acetonitrile); 20% B in 10 min to 100% B in 30 min and kept for 15 min) to yield 5 (1.0 mg). Pale yellow solid; HRMS: m/z [M–H]− = 246.0770 ; NMR data see Supplementary Table The ethyl acetate extract (1.5 L) of a S. albus::pNT42/pHY42 was extracted with ethyl acetate (3 × 2 L). The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The extract was subjected to silica gel column chromatography  using a dichloromethane/methanol gradient. Three fractions containing 6 were combined and subjected to preparative RP-HPLC , B (100% acetonitrile); 1% B in 10 min to 100% B in 30 min and kept for 10 min) to yield crude 6 (18.9 mg). The crude product was subjected to semipreparative RP-HPLC , B (100% acetonitrile), 1% B in 10 min to 100% B in 30 min and kept for 10 min) yielding deoxyneoaureothin (6) (7.7 mg). Yellow solid; HRMS: m/z [M + H]+ = 464.2426 ; NMR data see Supplementary Table A 2-L culture of S. albus pHY134 (nor#_PKS) culture was filtered through Celite 545, and the water phase was again extracted with ethyl acetate (2 × 4.5 L). The combined extracts were dried over anhydrous sodium sulfate, concentrated under reduced pressure and subjected to chromatography on silica gel . Further purification was achieved by preparative RP-HPLC , B (83% acetonitrile); 40% B in 5 min to 100% B in 25 min and kept for 15 min) to yield crude 9. This fraction was further subjected to semipreparative RP-HPLC  using an isocratic system 83% acetonitrile/water containing 0.1% trifluoroacetic acid (80:20) to yield 9 (3.5 mg). NMR and MS data see Supplementary Figs. 8 and 10 was subjected to preparative RP-HPLC , B (83% acetonitrile); 20% B in 10 min to 100% B in 30 min and kept for 15 min) to yield crude 8 and 10, respectively. Crude 10 was further purified by semipreparative RP-HPLC  to yield 10 (5.5 mg). NMR and MS data see Supplementary Figs. 8 was further purified by semipreparative RP-HPLC  to yield a mixture containing 8. This fraction was further subjected to semipreparative RP-HPLC  to yield 8 (0.8 mg). NMR and MS data see Supplementary Figs. 7 was subjected to preparative RP-HPLC , B (83% acetonitrile); 40% B in 5 min to 100% B in 25 min and kept for 15 min) to yield crude 7. Final purification was achieved by another semipreparative RP-HPLC  to yield 7 (2.7 mg). NMR and MS data see Supplementary Figs. 8 to 10, these compounds rapidly degraded after NMR measurements.All isolation steps were carried out in the dark to avoid the degradation of polyketides. The (emulsion-forming) ethyl acetate extract (4.5 L) of a Further information on research design is available in the Supplementary InformationReporting SummarySource Data file


Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. Although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. Engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. In this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (TGEV) genome, expressing a chimeric spike protein from a virulent United States (US) PEDV strain. This virus (rTGEV-RS-SPEDV) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. In addition, the virus caused very minor tissue damage compared with a virulent virus. The rTGEV-RS-SPEDV vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent PEDV US strain (PEDV-NVSL). The rTGEV-RS-SPEDV virus protected against challenge with a virulent PEDV strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus RNA levels in feces. The rTGEV-RS-SPEDV virus induced a humoral immune response specific for PEDV, including neutralizing antibodies. Altogether, the data indicated that rTGEV-RS-SPEDV is a promising vaccine candidate against virulent PEDV infection. Acute infectious diarrhea is a major cause of high morbidity and mortality in piglets worldwide. Enteric infections in animals are frequently associated with viruses, including rotaviruses and coronaviruses (CoVs) . A metagPedacovirus of genus Alphacoronavirus, included in the family Coronaviridae into the order Nidovirales .Baby hamster kidney cells stably transformed with the gene coding for porcine aminopeptidase N (BHK-pAPN)  were groTTAATTAAGAAGGGTAAGTTGCTCATTAGAAATAATGGTAAGTTACTAAACTTTGGTAACCACTTCGTTAACACACCATGAAGTCTTTAACCTAC-3′, PacI restriction site in italics) and PEDV-S-3952-RS (5′-CCACACATACCAAGGCCACTTGATGTATGTCTC-3′). TGEV sequence encoding S protein transmembrane domain and endodomain was amplified using pGEM-T-TGEV-SC11 intermediate plasmid as a template and oligonucleotides TGEV-S-4165-VS (5′-GAGACATACATCAAGTGGCCTTGGTATGTGTGG-3′) and TGEV-S-4439-RS . In all cases, PEDV sequences are indicated underlined. The obtained 4073 bp and 308 bp PCR products were used as templates and amplified with oligonucleotides Pac-S-VS and TGEV-S-4439-RS. The 4348 bp PCR product was digested with PacI and MluI and cloned into the same sites of pGEM-T-TGEV-SC11 plasmid, generating pGEM-TGEV-SPEDV plasmid. This plasmid was digested with PacI and MluI restriction enzymes and the fragment containing chimeric S gene was cloned into the same sites of pBAC-TGEV-SC11-P-M or pBAC-TGEV-SC11-RS .Antibodies induced against TGEV and PEDV viruses were detected by ELISA as described before . Briefly50 in the presence of medium or in the presence of serum sample.Heat-inactivated sera were incubated for 1 h at 37 °C in the presence of 100 pfus of PEDV virus in DMEM containing 5% FCS. Serial dilutions of the mixtures were added to confluent Vero cells in 96-well plates. After one hour of incubation at 37 °C, medium was removed and infection medium was added. After 72 h, cells were fixed with 10% formaldehyde in PBS, stained with a crystal violet solution, and virus titer was determined. The neutralization index was defined as the logarithm of the ratio of TCIDTotal intracellular RNA was extracted from Vero cells or tissue samples. Total RNA was purified with RNeasy Mini kit , according to the manufacturer’s specifications. To isolate viral RNA from fecal swabs, the swab was resuspended in 300 µL of PBS with antibiotics . RNA was isolated from 140 µL of that solution using QIAamp Viral Mini kit (Qiagen) following the manufacturers’ instructions. In all cases, total cDNA was synthesized using 100 ng of total RNA as a template, random hexamers, and the High-capacity cDNA transcription kit (Life Technologies), following the manufacturers’ recommendations.The viral genome region from 3b gene to 3′ UTR (nt. 25856–28544) was sequenced in all cases. To that end, overlapping PCRs were performed using oligo pairs described in Ct−ΔΔ method [PEDV genomic RNA (gRNA) levels were measured by RT-qPCR analysis using a custom TaqMan assay detecting a conserved ORF1a region . TGEV gRNA was evaluated using a custom TaqMan assay set up in our laboratory . The port method . All expt method .t tests were used to analyze the difference in mean values between groups. All results were expressed as means ± the standard deviations of the means. p values <0.05 were considered significant.Two-tailed, unpaired Student A chimeric S protein (SPEDV-TGEV) was designed. The N terminus of the protein contained the exposed domain of PEDV S protein from a virulent US strain A, includ6 TCID50/animal. Interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at 2 and 4 dpi , vaccination elicited PEDV-specific antibodies to a higher level in PEDV-NVSL vaccinated animals than in rTGEV-RS-SPEDV vaccinated ones A. This rTo further analyze the humoral response specific for PEDV, the levels of PEDV-specific IgGs B and IgAThe neutralization capability of the antibodies elicited was evaluated. As expected, non-vaccinated animals only induced the production of neutralizing antibodies after challenge . In contAltogether, the data indicate that rTGEV-RS-SPEDV virus conferred protection against PEDV infection in the young pig model and could be the basis for an effective vaccine candidate.An attenuated chimeric rTGEV virus expressing the ectodomain of a virulent US PEDV S protein (rTGEV-RS-SPEDV) was engineered as vaccine candidate for PEDV and evaluated in a young piglet model system. The rTGEV-RS-SPEDV virus elicited a neutralizing humoral response specific for PEDV. In fact, vaccinated animals were protected against challenge with a virulent PEDV virus.The attenuated live vaccine candidates are the best option for PEDV vaccination because non-replicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection ,26. TradIt is worth noting that in our rTGEV-RS-SPEDV vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. In contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome introduced during passages in cell cultures that, after passages in vivo, may lead to vaccine virus reversion to virulence.The engineered rTGEV-RS-SPEDV vaccine candidate has the advantage of replicating in the jejunum of infected piglets as efficiently as a virulent control virus, without causing significant pathology, and most likely leading to a protective IgA response. Indeed, vaccinated animals were protected against virulent PEDV challenge. In contrast, traditionally attenuated vaccines, as a consequence of adaptation to cell culture, have a dramatically reduced replication in the enteric tract  and, as Recently, a swine enteric coronavirus (SeCoV) that is a recombinant between TGEV and PEDV has been described in Europe ,68,69. TThe rTGEV-RS-SPEDV vaccine candidate was genetically stable in tissue culture; nevertheless, it evolved in vivo. Therefore, its biosafety could be improved by adding an additional safety guard at the 5′-end of PEDV virus genome (in the replicase gene), at a distal position from the attenuating mutations introduced by duplicating sequences closed to M, N and 7a genes  71,72,7,74. The


Drosophila melanogaster known as germ cell–expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand–binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in  Drosophila melanogaster ancestral paralog, germ cell–expressed (Gce) ,ed (Gce) –7. Met ad (Gce) –. bHLH-PAd (Gce) –. This apd (Gce) –, 11, andd (Gce) –, 12–14, Gce) –12–, 16.Kr-h1)  of Met/Gce , 17, 18 Kr-h1) , 20–24, squitoes , 18. To squitoes , 26.JH signaling clearly is vital to arthropods and important for our capacity to control insect pests and disease vectors by means of JH mimics. However, very little is known about how natural JHs or their synthetic agonists interact with the intracellular receptor and what decides which compound will or will not activate it.e.g. moths) differs from JH III by the presence of ethyl instead of methyl groups at the C7 and C11 carbons of the sesquiterpenoid backbone . The natural JH III with its single chiral center is the 10R enantiomer  cell line, we have previously shown that MF and JH III  induce a JH-responsive luciferase (JHRE-luc) reporter in a manner dependent on binding to the endogenous JH receptor Gce (supporting information). The effective concentration (EC50) values for inducing the JHRE-luc reporter were roughly in the 100–200 nm range for R,S-JH III and JHB3, whereas MF was about 20–40-fold less potent (A). RNAi-mediated knockdown of Gce revealed that, like MF and JH III .JH III, its precursor MF, and JHB3  are all nogaster , 32, 47.ptor Gce . Here, ws potent A. RNAi-md JH III , JHB3 aliK) of the ligands in competition against R,S-[3H]JH III. Although unlabeled R,S-JH III and JHB3 were both similarly more effective than MF in activating JHRE-luc in the S2 cells (A), JHB3 bound Gce with a higher iK (83.3 ± 40.8 nm) than did R,S-JH III (11.0 ± 2.2 nm) (B). Rather, the binding affinity of JHB3 to Gce was close to the iK established for MF (87.9 ± 22.2 nm) (B). To address the discrepancy, we tested the hormones using an independent two-hybrid assay in a human (HEK293T) cell line where ligand binding to Gce is estimated from JH-dependent interaction between Gce and its partner protein, Tai . Why JHB3 was relatively more active in the Drosophila S2 cells than in the two ligand-binding assays is unclear, but it might be due to differences in solubility or stability of JHB3 in the different experiments.To assess direct binding of the hormones to the Gce protein, we measured the inhibition constants  B. Rather22.2 nm)  Fig. 2BBKi of thein, Tai . This exDrosophila JH receptor Gce, albeit with different efficiencies. When compared with closely related compounds lacking JH activity, such as the JH precursor farnesol -JH I, which represents the native JH I configuration , efficiently competed against R,S-[3H]JH III for binding to the Gce protein, reaching a iK of 13.8 ± 5.1 nm, similar to that of R,S-JH III (B). In the independent two-hybrid assay, JH I and R,S-JH III were also equally effective in stimulating the interaction between Gce and Tai (C). As expected based on these data, JH I activated the JHRE-luc reporter in the S2 cells with an EC50 that was not significantly different from the activity of R,S-JH III or JHB3 in the same assay (A).To test whether Gce can recognize a juvenile hormone of insects other than ype JH I . SynthetS-JH III B. In the and Tai C. As expme assay A.Drosophila Gce is a functional receptor not only for the three endogenous JHs of the fly but also for the distinct lepidopteran JH I. The higher degree of epoxidation in JHB3 or the extra two carbons at the side chains of the JH I skeleton . At 1 μm concentration, R-JH III induced JHRE-luc expression more than 2-fold higher relative to S-JH III .Although the insect corpora allata glands almost exclusively synthesize the 10f JH III , 50, comS-JH III  in the Jturation A. At 1 μS-JH III A, inset.R-JH III was reflected in direct ligand binding to the Gce protein. Indeed, Gce displayed an 8-fold higher binding affinity (iK = 4.8 ± 1.3 nm) to the natural R enantiomer relative to S-JH III (iK = 38.3 ± 5.2 nm) in the competition assay against R,S-[3H]JH III (B). The preference of Gce toward R-JH III, albeit less pronounced, was confirmed in the two-hybrid assay in HEK293T cells, which revealed that R-JH III (EC50 = 73.1 ± 5.2 nm) was ∼2.3-fold more effective than S-JH III (EC50 = 169.8 ± 5.5 nm) in stimulating the Gce–Tai interaction (C).We next tested whether the stronger activity of H]JH III B. The preraction C.R-JH III enantiomer should be a better agonist than its optical antipode. However, the differences were relatively minor as even S-JH III attained appreciable activities in all assays (A–C), suggesting that the exact configuration at the epoxide group is not a critical requirement for JH III to activate its intracellular receptor.The three independent cell-based and ligand-binding assays yielded congruent data, supporting the expectation that the natural l assays , A–C, suR,11S-JH I, assuming the native 2E,6E and all three alternative E/Z configurations, hereafter referred to as S--JH I, S--JH I, and S--JH I, respectively (supporting information). In addition, we tested the altered double-bond geometry using isomers derived from the unnatural 10R,11R absolute configuration of JH I, designated R--JH I, R--JH I, and R--JH I (supporting information for NMR data). To assess the activities of the individual JH I geometric isomers as JH receptor agonists, we determined five parameters for each isomer, namely (i) affinity of binding to the Gce protein in vitro, (ii) ligand-induced interaction between Gce and Tai in the two-hybrid assay, (iii) activation of the JHRE-luc reporter in S2 cells, (iv) transcriptional induction of the JH-response Kr-h1 gene in vivo, and (v) the capacity to affect Drosophila development.To address the importance of configuration of the double bonds in the JH backbone, we used a set of synthetic geometric isomers of 10B), the native S--JH I isomer bound avidly to Gce . Altering the configuration at either one of the double bonds in JH I reduced this affinity ∼32- and 45-fold, respectively, as determined in the same binding assay for the S--JH I and S--JH I isomers -JH I no longer efficiently competed against R,S-[3H]JH III in Gce binding; its apparent affinity dropped about 1640-fold (iK = 22.6 μm) relative to S--JH I  previously reported for the biologically inactive JH precursor farnesol  ≫ E,Z > Z,E ≫ Z,Z.As already shown above B, the naS--JH I elicited this interaction with an EC50 of 53.4 ± 13.9 nm, whereas S- and S--JH I ranked second and third by being ∼23- and 30-fold less effective; the S--JH I isomer was inactive (B).Similar data were obtained from the two-hybrid assay of JH-induced Gce–Tai interaction. The native inactive B.Drosophila S2 cell–based assay where activation of the JHRE-luc reporter by JH I depends on Gce . While native S--JH I was by far the best activator, comparable with R,S-JH III (A), the S-- and S--JH I isomers induced strong JHRE-luc expression only at the highest, 10 μm dose, and S--JH I was ineffective even at this concentration . Because JHRE-luc activation by the unnatural geometric isomers did not reach saturation, rather than EC50 we compared values at 1 μm concentration where S-- and S--JH I achieved moderate induction (C).Importantly, the same ranking was revealed in the S-JH III A, the S-nduction C.R-, R-, and R- geometric JH I isomers with the 10R,11R configuration - from the second ranking R--JH I, the iK values of the 10R,11R isomers matched those determined for the corresponding 10R,11S homologs remarkably well -JH I isomer performed better than R--JH I in the transcriptional activation of JHRE-luc in S2 cells . Although we did not have the 10R,11R optical isomer of -JH I to directly assess the effect of the absolute configuration at the C11 chiral center, comparison of the Gce binding and transactivation data, particularly those obtained for R--JH I and S--JH I, suggests that, while the natural JH I enantiomer is a better agonist, the chirality is of secondary importance relative to the double-bond geometry.To further corroborate these results, we tested guration . Their bbly well . Consistin vitro binding and the cell-based assays agree with an authentic transcriptional response to JH, we chose the Kr-h1 gene, which is directly induced by the ligand-activated JH receptor complex -JH I hormone followed by the S--JH I and R--JH I isomers that were 5- and 7.2-fold less effective, respectively (A). Alteration of the C2 double bond was more detrimental, leading to 14- and 34-fold reduction of activity, respectively, of the S--JH I and R--JH I isomers. Both S--JH I and R--JH I were virtually inactive, retaining less than 1% of the activity of the natural JH I (A). These in vivo data thus perfectly agreed with the capacity of the geometric isomers to bind the Gce protein and to activate the JHRE-luc reporter in Drosophila S2 cells -JH I was about 5-fold more potent in preventing normal adult development than the second ranking S--JH I (B). This result exactly matched the capacity of these two isomers to induce Kr-h1 transcription in the treated pupae (A). By contrast, activity of the R--, R--, and S--JH I isomers was more than 100-fold weaker compared with S--JH I. The two remaining S-- and R--JH I compounds were essentially ineffective, causing failure to eclose in 32 and 17% adults, respectively, only at the highest tested dose (B). These results indicated that altering the double-bond geometry at either the C2 or both the C2 and C6 positions rendered JH I inactive in the white puparium bioassay.Application of JH or its mimics to white puparia of elopment , 53, 54.,Z)-JH I B. This red pupae A. By conted dose B. These Tribolium castaneum . The 3D model revealed an α/β-fold with a unique α-helix between amino acids Asp-311 and Cys-325 in the Drosophila Gce protein (Asp-50 to Cys-64 of the modeled region), matching a homologous helix in Tribolium Met. The results indicate that JH receptors from both species share a similar structural fold . The model of the Gce hormone–binding pocket was extended to dock the native JH III and JH I and the geometric isomers of JH I in both the normal 10R,11S and the unnatural 10R,11R absolute configurations. The docking identified bound states for all ligands with highly similar conformations within the binding pocket (A). The ΔG of binding for each ligand was determined using the MMGBSA  approach , ranking the JH I ligands in the expected order with the most negative ΔG for the native hormone and increasing to the least negative values for the inactive Z,Z isomers .To address the selectivity and binding mechanism of the JH receptor, we modeled the 3D structure of the Gce PAS-B domain and compared it with that of the PAS-B domain previously modeled for the Met ortholog from the beetle astaneum . The strg pocket A. The ΔG isomers . The rel of JH I B.S--JH I, the calculated strain energies were the lowest for JH III and JH I in their natural conformations and the highest for the inactive Z,Z isomers  (B). To test the contribution of this hydrogen bond to JH binding by the Gce protein in vitro, we removed the hydroxyl group by mutating Tyr-270 to phenylalanine. The GceY270F mutant retained 40% of the [3H]JH III binding capacity of the WT Gce protein (C). Consistent with this reduction, MF that lacks the epoxide and thus cannot form the hydrogen bond with Tyr-270 displayed a lower binding affinity to WT Gce relative to the affinity of JH III and was also less effective than JH III in inducing both the JHRE-luc reporter and the ligand dependent Gce–Tai interaction (A–C).The position of the docked JH ligands in our Gce PAS-B model corresponds to the reported model of the  complex . Both mo region) B. To tes protein C. Consiseraction , A–C.supporting information). With a 143.4 ± 45.0 nmiK assessed from competition against R,S-[3H]JH III, deoxy-JH I bound the Gce protein with an affinity about 10-fold lower than that of S--JH I (A). At 1 μm concentration, deoxy-JH I activated JHRE-luc in S2 cells to ∼17% of the level achieved with the native JH I (C). Consistent with these data, deoxy-JH I showed intermediate activities in inducing both the Kr-h1 mRNA expression and developmental arrest in Drosophila pupae (A and B).To examine the impact of epoxidation loss on Gce binding to JH I, we used a compound that represents a nonepoxidated version of JH I, herewith referred to as “deoxy-JH I” -JH I A. At 1 μive JH I C. Consisla pupae , A and BJHRE-luc reporter in Drosophila S2 cells  and thus closer to the affinity of the native JH III or JH I (A). Both fenoxycarb and W330 were more effective than the native JHs in inducing the ligand-dependent interaction of Gce with Tai (B). The carbamates were also much stronger activators of the JHRE-luc reporter in S2 cells, reaching EC50 values of 2.3 ± 0.7 and 5.4 ± 0.4 nm for fenoxycarb and W330, respectively (C). RNAi knockdown experiments confirmed that, like the native JHs, fenoxycarb and W330 activated JHRE-luc expression through Gce rather than the alternate JH receptor Met .Here, we tested agonist capacities of another type of compounds, the bicyclic carbamate derivatives, represented by the insecticide fenoxycarb and a carbamate juvenoid, W330  Fig. 1)Fig. 1).  or JH I A. Both fwith Tai B. The caectively C. RNAi kin vivo, and particularly fenoxycarb was at least as effective as JH I in inducing either Kr-h1 mRNA expression or the morphogenetic defects in Drosophila pupae (D and E). These data indicate that despite their chemistry, unrelated to that of JH, the synthetic carbamates are potent JH receptor agonists.Consistent with the above results, both carbamates were highly active la pupae , D and EAn intracellular receptor for JH had long remained elusive, which caused a major gap in our knowledge of how this versatile hormone directs insect life. Now that the receptor proteins are known, we have begun to bridge this gap by testing which structural features are critical for a JH to exert its agonist activity.D. melanogaster, namely JH III, JHB3, and MF, bind Gce and activate its transcriptional response. Compared with JH III, the other two hormones, JHB3 and MF, have lower affinity to Gce, which might be compensated by higher titers of both JHB3 and MF in the hemolymph .The Gce receptor could not discriminate, in any of our assays, between the native pression . The prepression  increaser JH III . Nonether JH III  of the GB domain C.R-JH III enantiomer, which is synthesized by the insect endocrine glands  affinity of 10R-JH III for a hJHBP and its 12-fold greater biological activity in lepidopteran species  geometric isomer of JH I was only 2.5–3.5-fold more effective than its antipode in blocking metamorphosis in diverse insects   –46. Simi hormone . TogetheBombyx mori silkworms that cannot synthesize epoxidated JHs due to deficiency of a JH epoxidase pupate prematurely, suggesting that JH precursors such as MF cannot sustain normal development of B. mori -JH I hormone in all assays that reflect either Gce binding or transcriptional activation. Therefore, the absence of epoxide either from JH III (MF) or from JH I generally lowers the agonist activity by about 1 order of magnitude.The epoxide ring connecting carbons 10 and 11 has always been considered an important functional feature of native insect JHs. Consistently, mutants of the  B. mori . However hormone , 56. MF  and Gce  or their and Gce  and in tB. mori and the epoxide oxygen of JH III. Interestingly, a hydrogen bond was also found between the JH III epoxide and Tyr-129 in a crystal structure of an otherwise heterologous mJHBP of the mosquito Aedes aegypti  The native 2E,6E conformation is the most active in all tests. 2) A single “twist” in the JH backbone markedly reduces all agonist activities. 3) Geometry is more critical at the 2,3 double bond than at the 6,7 double bond. 4) Altering the geometry at both double bonds renders an isomer inactive. It is gratifying to see that these rules apply, without exception, in the five diverse assays we performed with these compounds.Ligand-binding, cell-based reporter, and Rhodnius prolixus, the geometric isomers retained 7.3 , 3.3 , and 0.2%  of the potency of the native E,E conformation of 10R,11S-JH I -JH I was the strain energy similar to that calculated for the JH I and JH III native hormones, but S--JH I apparently lost preferential noncovalent bonds to the protein cavity.To address the problem more precisely, additional aspects have to be taken into account. For instance, rather than docking scores, one should attempt to determine Gibbs free energy of binding, which involves ligand internal flexibility and solvation/desolvation effects. While in the binding pocket, all ligands assumed similar conformations that did not correspond to their global energy minima. Based on the structural model of the binding and the calculated strain energies, we can conclude that the internal conformational barriers between the bound and unbound states of the ligands present a major factor discriminating between productive and nonproductive binding modes. If the difference between ligand conformations in bound and unbound states is large while most of the ligand–protein interactions are sustained, it is clear that Gibbs free energy of binding will primarily be determined by this difference. Only in one case of the moderately active stereoisomer In summary, our present work reveals that the intracellular JH receptor Gce displays stereoselectivity toward its hormone agonists. Of greatest importance is the geometry of the JH skeleton where alteration is most critical at the 2,3 and better tolerated at the 6,7 double-bond position. Of moderate importance is the presence of the C10,C11 epoxide followed by the optical isomerism at the C10 and C11 positions. Contrasting with this exquisite stereoselectivity is the fact that Gce recognizes chemically disparate compounds such as the pyridine derivative pyriproxyfen  was purchased from PerkinElmer Life Sciences. R,S-JH III and fenoxycarb (ethyl N-[2-(4-phenoxyphenoxy)ethyl]carbamate) were from Sigma-Aldrich; methyl farnesoate  was obtained from Echelon Biosciences. The enantiomers of JH III (10R and 10S), separated using HPLC on a chiral stationary phase (supporting information). A series including the natural configuration of JH I with its stereoisomers -7-ethyl-3,11-dimethyltrideca-2,6,10-trienoate (a nonepoxidated version of JH I) were a generous gift from Dr. Karel Sláma; we verified their identity and purity using NMR (supporting information). Ethyl N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (referred to as W330) was kindly provided by the author of the compound synthesis, Dr. Zdeněk Wimmer .Racemic ry phase , were kioisomers  and methk Wimmer   with an N-terminal Myc epitope was expressed by in vitro transcription/translation from a codon-optimized DNA template in the pK-Myc-C2 plasmid using the rabbit reticulocyte lysate TnT Quick T7 Coupled System (Promega) as described previously , 5 mm magnesium acetate, 1 mm EDTA (pH 8), 1 mm DTT), and the 100-μl reaction was incubated at room temperature for 1 h. For competition assays, a constant amount of R,S-[3H]JH III was combined prior to protein addition with increasing input (0.0003–300 pmol) of a cold competitor.The R,S-[3H]JH III bound against the molar concentration of a competing ligand, and iK values were calculated as described below (see “Data processing and statistics”).For the competition assays, we modified a method previously used for the ecdysone receptor . BrieflyR,S-[3H]JH III–binding assays and to complement data obtained with the above glass filter method, we used adsorption of unbound ligand to dextran-coated charcoal (DCC) (m Tris-HCl (pH 7.5), 1.5 mm EDTA, 1% dextran, 5% Norit A) was added to the 100-μl binding reaction, incubated for 2 min, and centrifuged for 3 min at 12,000 × g. The supernatant (100 μl) was collected for scintillation counting. The glass filter and the DCC methods yielded fully consistent results.For some al (DCC) , which wal (DCC) , 6. In tD. melanogaster S2 cells were grown at 26 °C in Shields and Sang M3 insect medium (Sigma-Aldrich) supplemented with 0.5 g/liter KHCO3, 8% fetal bovine serum (Life Technologies), 100 units/ml penicillin, and 100 μg/ml streptomycin. The assay was based on a JH-inducible firefly luciferase reporter (JHRE-luc) driven by eight tandem repeats of a JHRE from the A. aegypti early trypsin gene (JHRE-luc (125 ng/well) and plasmids encoding the Renilla luciferase (50 ng/well) and the D. melanogaster Tai protein (125 ng/well) using FuGENE HD DNA transfection reagent (Promega). For RNAi experiments, double-stranded RNAs (1 μg/well) targeting either gce, Met, or tai mRNAs (egfp for control) were prepared as described previously .sin gene  and was D. melanogaster proteins Gce, Met (both in pACT), and a C-terminally truncated Tai (in pBIND) and reproduced the assay in the human HEK293T cell line. The HEK293T cells were grown at 37 °C under a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells seeded in 24-well plates were transfected using FuGENE HD (Promega) with a 200 ng/well pG5luc reporter and 50 ng/well pACT-gce (or pACT-Met) and pBIND-Tai, which also encodes Renilla luciferase. Thirty-six hours post-transfection, cells were treated with a JH agonist in ethanol and incubated for another 8 h before EC50 values were determined from the relative luciferase/Renilla activity as described below (see “Data processing and statistics”).An assay that measures ligand binding–dependent dimerization of JH receptor proteins with their partner Tai was recently developed by Miyakawa and Iguchi  based onD. melanogaster at the white puparium stage; controls received 0.25 μl of acetone only. Twelve white puparia were used per treatment, and 24 h later they were pooled by three to obtain four replicates representing each treatment. The puparia were immediately frozen and subjected to total RNA extraction using TRI Reagent (Sigma-Aldrich) following the manufacturer's protocol. RNA was treated with TURBO RNase-free DNase (Ambion), and 2-μg RNA aliquots were used for first-strand cDNA synthesis using the SuperScript III kit (Invitrogen) with random hexamer primers. Transcripts were quantified using a LightCycler 480 qRT-PCR System (Roche Applied Science) with SYBR Green fluorescent label and previously described primers specific for Kr-h1 and the ribosomal protein 49 (rp49) genes  using a Burkhard microapplicator on 9) genes . All bioin vivo, we used the established white puparium bioassay ); Y = Bottom + (Top − Bottom)/(1 + 10 (X − log EC50)). The dK of [3H]JH III binding to Gce was previously established as 19.27 nm  with the “sigmoidal dose-response (variable slope)” equation: Y = Bottom + (Top − Bottom)/(1 + 10 ((log EC50 − X) × Hill slope)). The values are given in the corresponding figure legends.Results were plotted, and all the statistics were calculated using the Prism graphic program . In competition ligand-binding assays, D. melanogaster Gce PAS-B protein sequence was retrieved from the UniProt database (UniProt accession number Q9VXW7). The model was created with the homology module of MOE software (http://www.chemcomp.com)TM code 3F1P) (T. castaneum Met PAS-B (–G bind(NS) = MMGBSA ΔG bind − Receptor strain − Ligand strain where MMGBSA ΔG bind = Complex − Receptor − Ligand. The important parameter is the Prime MMGBSA ligand strain energy. To obtain this value, the ligand was extracted from the optimized complex, and an energy calculation was run on it without minimization to find the energy of the ligand as optimized in the binding pocket. Next, energy minimization was run on the ligand outside of the receptor. Both calculations were done on the ligand alone in solution. The energy difference is the ligand strain energy.The software  (http://de 3F1P)  as the tet PAS-B . The proet PAS-B –68. The t PAS-B –. The strt PAS-B –. The lowt PAS-B –, 6. The t PAS-B –. The binL. B., P. J., J. V., R. H., and M. J. conceptualization; L. B., R. H., and M. J. funding acquisition; L. B., P. J., M. D., P. K., J. V., R. H., and M. J. investigation; L. B., P. J., M. D., P. K., J. V., and M. J. methodology; L. B., R. H., and M. J. writing-original draft; P. J., M. D., and M. J. validation; J. V., R. H., and M. J. supervision; R. H. and M. J. resources; R. H. and M. J. writing-review and editing; M. J. project administration.


MT1F, MT1G, MT1L, and MT1X) predicted survival better than any combination of two-, three-, four-, five-, or six-gene models. In conclusion, this study is the first to report that simultaneous downregulation of six MT mRNAs’ levels in CRC patients, and their aberrant expression together, accurately predicted CRC patients’ outcomes.Colorectal cancer (CRC) is a heterogeneous disease resulting from the combined influence of many genetic factors. This complexity has caused the molecular characterization of CRC to remain uncharacterized, with a lack of clear gene markers associated with CRC and the prognosis of this disease. Thus, highly sensitive tumor markers for the detection of CRC are the most essential determinants of survival. In this study, we examined the simultaneous downregulation of the mRNA levels of six metallothionein (MT) genes in CRC cell lines and public CRC datasets for the first time. In addition, we detected downregulation of these six MT mRNAs’ levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. In order to understand the potential prognostic relevance of these six MT genes and CRC, we presented a four-gene signature to evaluate the prognosis of CRC patients. Further discovery suggested that the four-gene signature ( APC, p53, KRAS, and BRAF) are used to detect CRC, but these biomarkers are not sufficiently sensitive and specific. Thus, there is an urgent need for the identification of efficacious biomarkers, therapeutic targets, and agents for early diagnosis, prevention, and personalized therapy in CRC [Colorectal cancer (CRC) is the third most frequent tumor-related cause of mortality of men and women worldwide . The numMT1A, -B, -E, -F, -G, -H, -M, and -X. The rest of the MT1 genes  are pseudogenes whose protein product has not been found in humans [With the development of high-throughput technology, gene expression profiles have been broadly used to identify more novel biomarkers . Howevern humans . Numeroun humans ,14. AlthThus, in the present study, simultaneous downregulation of six MT mRNAs’ levels was examined in CRC cell lines and public CRC datasets. In addition, we also found downregulation of the six MT mRNAs’ levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. Moreover, we further investigated the prognostic significance of these factors for CRC outcomes.http://discover.nci.nih.gov/cellminer) to determine the mRNA expression of MTs in NCI-60. MT1B, MT1F, MT1G, MT1H, MT1L, and MT1X, respectively. In most of the datasets, mRNA levels of the six MT genes were decreased in most of the tumors, as opposed to in normal tissues. The most notable among these tumors was CRC, which showed the greatest number of cases of decreased expression levels of MT genes. In CRC cases, decreased expression levels of MT genes were observed in a total of 11 datasets for MT1B, 24 datasets for MT1F, 26 datasets for MT1G, 28 datasets for MT1H, 9 datasets for MT1L, and 25 datasets for MT1X. Taken together, these results indicate that mRNA expression levels of MT genes are downregulated in various cancers, especially in CRC.Our recent study showed that the metallothionein (MT) gene was one of the most significantly downregulated genes in CRC clinical tissues compared with normal colorectal tissues by analysis of a Gene Expression Omnibus (GEO) dataset GSE21815) (our unpublished data from reference ) Supple. We foun (our unpMT1B was downregulated in about 87% (26/30) of CRC tumor tissues (MT1F was downregulated in about 87% (26/30) of CRC tumor tissues (MT1G was downregulated in about 70% (21/30) of CRC tumor tissues (MT1H was downregulated in about 77% (23/30) of CRC tumor tissues (MT1L was downregulated in about 90% (27/30) of CRC tumor tissues (MT1X was downregulated in about 80% (24/30) of CRC tumor tissues  and adjacent non-tumor (N) CRC specimens. The results showed that  tissues A; MT1F w tissues B; MT1G w tissues C; MT1H w tissues D; MT1L w tissues E; and MT tissues F. Taken MT1B (a fold change of −7.717) (MT1F (a fold change of −6.946) (MT1G (a fold change of −10.010) (MT1H (a fold change of −9.898) (MT1L (a fold change of −9.108) (MT1X (a fold change of −5.364) (n = 101) compared to normal colon tissues (n = 19). These results suggest that MT genes are downregulated in CRC and may be attractive markers for the diagnosis of CRC.Further, to confirm the expression levels of the six MT genes in a large number of CRC tissues, we analyzed mRNA expression profiles of six MT genes using existing complementary DNA (cDNA) microarray datasets deposited in the Oncomine database. In the TCGA microarray dataset of the Oncomine database with colorectal tumor and normal colorectal tissues, significant decreases were found in the mRNA expression of  −7.717) A, MT1F ( −6.946) B, MT1G (−10.010) C, MT1H ( −9.898) D, MT1L ( −9.108) E, and MT −5.364) F in CRC SurvExpress survival analysis [n = 350) were classified into predicted low- and high-risk groups according to the prognostic index (PI)  . The clilow risk . Surviva of MT1B A, MT1H  of the 350 patients was from −9.881 to −7.151, with an optimal cut-off value of −8.134. Those with PI less than −8.134 were placed into the low-risk group (n = 195), while those with PI higher than −8.134 formed the high-risk group (n = 155). The analysis demonstrated that low risk was correlated with high expression of MT1F, MT1G, MT1L, and MT1X, while high risk was correlated with low expression of MT1F, MT1G, MT1L, and MT1X  (n = 195) had significantly longer survival times than did those with a predicted high risk (n = 155) (p = 0.00351)  B. Moreov0.00351) C. Taken MT1F, MT1G, MT1L, and MT1X-four-gene signature combination is related to survival and is a predictor of prognosis in CRC patients. There are several lines of evidence that support this conclusion. First, we demonstrated simultaneous downregulation of the mRNA levels of six MT genes in CRC cell lines and public CRC datasets. Second, downregulation of the six MT mRNAs’ levels was detected in clinical NT pairs of CRC specimens. Third, we found that high expression of MT1B, MT1H, or MT1L was significantly correlated with good prognosis in CRC patients. Fourth, the most significant four-gene signature model was shown to be related to survival and a predictor of prognosis in CRC. Collectively, this study is the first to report simultaneous downregulation of six MT mRNAs’ levels in CRC patients and their aberrant expression together, accurately predicting CRC patients’ outcomes.CRC is a heterogeneous disease composed of biologically and clinically diverse diseases. This complexity causes the molecular characterization of CRC to remain deficient, with a lack of clear gene markers associated with CRC and to the prognosis of this disease ,20. Thusp53, KRAS, and BRAF [MT1B, MT1F, MT1G, MT1H, MT1L, and MT1X—were analyzed among the top 20 downregulated genes in CRC clinical tissues when compared with normal colorectal tissues by analysis GEO dataset (GSE21815) [Current molecular changes in colorectal tumors are usually linked to the traditional determination of somatic mutations in well-known tumor-suppressor genes or oncogenes, such as and BRAF . Moleculand BRAF ). Despitand BRAF . In thisSE21815) . FurtherMT1B, MT1F, MT1G, MT1H, MT1L, and MT1X single gene expression with the prognosis of CRC patients in the TCGA-CRC dataset from the SurvExpress database. The data demonstrated that low expression of MT1B, MT1H, or MT1L was significantly correlated with a high risk of poor prognosis (p < 0.05). However, the efficacy of a single index was limited. Therefore, multi-gene-combination prediction can improve the sensitivity to clinical outcomes of heterogeneous diseases such as cancer to mRNA abundance levels. Thus, we identified for the first time, the most significant four-gene signature model  that was able to predict survival and CRC prognosis. Overall, this multi-gene panel may serve as a promising outcome predictor and as potential therapeutic targets in CRC patients.The recent advances in genomic and transcriptomic technologies applied to the study of clinical samples have opened the way to obtaining genome-wide expression profiles of multiple patient cohorts and correlating the expression of certain genes with disease outcome ,26. ImpoIn conclusion, we consider that the results presented in this work provide strong support and a solid rationale for the exploration of changes in expression of MTs in CRC to assist in the development of clinically useful outcome prediction of CRC. Human 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens were obtained from the Department of Surgery, Taipei Medical University Hospital . Informed written consent was obtained from all patients and/or guardians for the use of their resected specimens. Acquisition of samples and their subsequent examination were approved by the Institutional Review Board (IRB) of Taipei Medical University (TMU-JIRB No.: 201312039). None of the participants had a previous history of cancer.Total RNA was extracted from NT pairs of CRC tissues using a Qiagen RNeasy kit  and Qiashredder columns according to the manufacturer’s instructions (Qiagen). One microgram of total RNA was reverse-transcribed to complementary (c)DNA using a Reaction Ready™ First Strand cDNA Synthesis Kit . To detect mRNA expression levels of MT genes and GAPDH, specific products were amplified and detected using the cycle profile of the Qiagen SYBR green reagent (Qiagen). The sequences of primers used in this study are listed as follows: MT1B: forward 5′-GCACCACAGGTGGCTCCTG-3′ and reverse 5′-TGGGCACACTTGGCACAGC-3′; MT1F: forward 5′-CAACTGCTCCTGCGCCGCT-3′ and reverse 5′-CCAGACCATGGAGATGGCC-3′; MT1G: forward 5′-CGCTGCAGGTGTCTCCTGC-3′ and reverse 5′-CAGCCCTGGGCACACTTG-3′; MT1H: forward 5′-CCTGCGAGGCTGGTGGCTC-3′ and reverse 5′-CTGGGCACACTTGGCACAG-3′; MT1L forward 5′-GCTCCTGCTCCTGTGCCAG-3′ and reverse 5′-TCTGGGAGCAGGGCTGTCC-3′; MT1X: forward 5′-GCTCGCCTGTTGGCTCCTG-3′ and reverse 5′-TGCAGATGCAGCCCTGGGC-3′; and GAPDH: forward 5′-AATCCCATCACCATCTTCCA-3′ and reverse 5′-TGAGTACGTCGTGGAGTCCA-3′.http://discover.nci.nih.gov/cellminer; version 1.5) was used to compare and plot the relative baseline expression of MT mRNA in the NCI-60 cell line panel. The tool enables retrieval and integrated analysis of baseline and experimental data compiled from the 60 cell lines included in the panel. CellMiner gene transcript data were generated from microarray platforms. We selected gene transcript level z-scores for analysis of the six MT genes as gene identifier inputs.The CellMiner tool . The threshold search criteria used in the study were a p-value of <0.001, a fold change of >2, and a gene rank in the top 5%.Gene expression changes were analyzed in the TCGA microarray dataset of the Oncomine website with colorectal tumor and normal colorectal tissues , which is a comprehensive gene expression database, can provide risk assessment and survival analysis in cancer datasets using a biomarker gene list as an input. The samples of each dataset were split into two risk groups with the same size; each group was determined according to the ordered prognostic index (PI) with the dataset split by the ordered PI  so as to have an equal number of samples in each group. The PI was computed using the expression levels and values obtained from the Cox fitting algorithm [In our analysis, lgorithm .p-Values and fold-changes for differential expression analysis of genes generated from NT pairs of CRC tissues and the Oncomine database were calculated using a one-sided Student’s t-test. p values of <0.05 were considered significant.


The A-alleles of rs3764261, rs4783961, and rs1800775 variants were found to be independently associated with 2.86, 1.71, and 0.91 mg/dL increase in HDL-C per allele, respectively. In addition, the A-allele of rs4783961 was significantly associated with a reduced T2D risk (odds ratio (OR), 0.82; 95% confidence interval (CI), 0.71–0.96)), and the A-allele of rs1800775 was significantly related to a lowered DKD risk . CETP expression was significantly decreased in the T2D mice kidney compared to that in the control mice . These collective findings indicate that CETP variants in the promoter region may affect HDL-C levels. Taiwanese individuals possessing an allele associated with higher HDL-C levels had a lower risk of T2D and DKD.Cholesteryl ester transfer protein (CETP) plays an important role in lipid metabolism. Low levels of high-density lipoprotein cholesterol (HDL-C) increase the risk of type 2 diabetes (T2D). This study investigated CETP gene variants to assess the risk of T2D and specific complications of diabetic kidney disease (DKD) and diabetic retinopathy. Towards this, a total of 3023 Taiwanese individuals  were enrolled in this study. T2D mice ( Type 2 diabetes (T2D) is a chronic metabolic disorder with a multifactorial pathogenesis. T2D involves complex interactions between environmental factors and genetic predisposition ,2. MacroThe characteristics of diabetic dyslipidemia include high serum triglyceride concentrations, low serum high-density lipoprotein cholesterol (HDL-C) levels, and elevated low-density lipoprotein cholesterol (LDL-C) levels. In several epidemiological studies, low levels of HDL-C were associated with an increased risk of T2D ,6. FactoIn the present study, we evaluated the respective effects of three single nucleotide polymorphisms (SNPs) located in the CETP promoter region—rs3764261, rs4783961, and rs1800775—and a missense coding SNP (rs5882) of the CETP gene on the following: lipid concentrations, associated risk of T2D, and the specific complications of diabetic kidney disease (DKD) and diabetic retinopathy (DR) in a Taiwanese population. In addition, we used a T2D mouse model to determine the expression levels of CETP in adipose, kidney, and retinal tissues.2 [1C (HbA1C), total cholesterol, HDL-C, LDL-C, triglycerides, and eGFR levels were determined. The non-diabetic controls consisted of 1383 subjects (age range 30–70 years) who were randomly selected from the Taiwan Biobank (TWB) [https://www.twbiobank.org.tw/new_web_en/index.php). The inclusion criteria for selecting controls in the association study were no history of T1D, diagnosis of T2D, and an HbA1C < 6.0%. Among these 1383 non-diabetic controls, 349 individuals aged ≥55 years and 253 individuals residing in Taichung and neighboring countries were selected as an age control group and a geographical region control group, respectively, to perform the association studies. The study was approved by the institutional review boards on CMUH, Taiwan (IRB No. CMUH103-REC2-071(CR-3)), and by the Ethics and Governance Council (EGC) of Taiwan Biobank, Taiwan. The information of non-diabetic controls was applied from Taiwan Biobank which is governed by the EGC and the Ministry of Health and Welfare, Taiwan. The individual genotype information could not be released due to the regulations but the summary statistics could be queried in the Taiwan View website (https://taiwanview.twbiobank.org.tw/index).The study involved 1640 unrelated individuals with T2D (age range 24–96 years), who were recruited from the China Medical University Hospital (CMUH), Taichung, Taiwan. Individuals with type 1 diabetes (T1D), gestational diabetes, or maturity-onset diabetes of the young, were excluded. Subjects were diagnosed using the American Diabetic Association Criteria . DKD was2 . Of this2 . Among tnk (TWB) , a natiohttps://www.ncbi.nlm.nih.gov/genome/guide/human/) and, based on previously published reports, have been associated with an effect on HDL-C levels [p < 0.05 indicated deviance from HWE). T2D subjects were genotyped using the Hap550K-BeadChip , which has been used previously for genome-wide association studies in the Han Chinese population of Taiwan [Three SNPs—rs3764261 (-2568A/C), rs4783961 (-998A/C), and rs1800775 (-629A/C)—located in the promoter region , as well as a missense coding SNP rs5882 (+16A/G) in exon 14  of the CETP gene were included. The SNPs were selected from the human genome database , and their non-diabetic littermates  were obtained from the National Laboratory Animal Center . Six male mice per group aged 32 weeks were used for the experiment. All mice were housed under light/dark conditions (12 h/12 h) with free access to water and food. Tail vein blood samples were used to monitor blood glucose levels every two weeks . All animal care and handling were approved by the Institutional Animal Care and Use Committee of China Medical University (CMUIACUC-2017-207-1).Six weeks-old T2D mice (BKS.Cg-w/v) sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.45 μm pore size nitrocellulose membranes . The membranes were incubated with anti-CETP primary antibody  overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (GeneTex) at room temperature for 1 h. Anti-β-actin  was used as an internal control. The blot signals were developed using an enhanced chemiluminescence system . The ImageJ program  was used to quantify protein expression, which was normalized to the expression of the internal control.Radioimmunoprecipitation lysis buffer  containing protease inhibitors and phosphatase inhibitors  was used to extract the proteins from mouse kidney and retina tissues. Protein extracts (20 μg) were separated using 12% (Mouse adipocyte tissue sections (5-μm) were deparaffinized and soaked in a 3% hydrogen peroxide solution in distilled water for 5 min to counteract endogenous peroxidase reactions. The sections were further incubated with primary antibody against CETP , followed by incubation with horseradish peroxidase-conjugated secondary antibody. For all sections, the presence of peroxidase was detected by the addition of 3, 3′-diaminobenzidine tetrahydrochloride solution, and finally counterstained with hematoxylin.t-test for continuous variables and chi-square test for categorical variables were used to compare the characteristics and clinical data of T2D subjects and controls. One-way analysis of variance (ANOVA) or general linear regression models were used to analyze the effect of the CETP gene SNP genotypes on lipid levels. Chi-square goodness of fit test was used to check compliance with the Hardy–Weinberg equilibrium. Association analysis was carried out to compare genotype distribution between T2D subjects and non-diabetic controls, or subjects with or without DKD, or subjects with or without DR, using additive models. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined by logistic regression and were adjusted for age, sex, HbA1C, or BMI levels. All statistical analyses were performed using the IBM SPSS Statistics 22 . A p-value of less than 0.05 was considered statistically significant. Haploview software (version 4.2) was used to estimate the frequencies of CETP haplotypes and to calculate the linkage disequilibrium (D’) between any two loci [The Student’s two loci .1C levels, lower BMI levels, lower systolic and diastolic blood pressure, lower waist–hip ratio, higher total cholesterol, higher HDL-C, lower LDL-C, lower triglycerides, and higher eGFR levels than did the T2D patients. Among the T2D patients, the mean duration of diabetes was 11.0 ± 8.7 years and 475 of the 1063 T2D patients were diagnosed with DR (18.9% with NPDR and 25.8% with PDR); further, of the 1325 of the 1640 T2D subjects for whom eGFR values were obtained, 27.7% (367/1325) had DKD.The study included 1383 non-diabetic controls and 1640 patients with T2D. The demographic and clinical characteristics of these groups are summarized in We estimated the change in HDL-C in the different genotypes of CETP SNPs. The A-alleles of rs3764261, rs4783961, and rs1800775 were independent of age and sex and were associated with a 2.86, 1.71, and 0.91 mg/dL increase in the levels of HDL-C per allele, respectively . We did We then analyzed the risk of CETP variants in patients with T2D and non-diabetic controls. As shown in 1C was associated with decreased risk of T2D with DKD in the additive model . The A-allele of rs1800775 was also independently associated with sex, age, HbA1C, and BMI was associated with decreased risk of T2D with DKD in the additive model . However, none of the studied CETP variants was associated with the risk of T2D in patients with DR.We subsequently compared the risk of CETP variants in the T2D patients according to the presence and absence of DKD  and with/without DR. As shown in Pairwise linkage disequilibrium analysis revealed a strong linkage disequilibrium in three of four SNPs  located in the CETP promoter region (D’ ≥ 0.945). As shown in p = 0.020, p = 0.467, The T2D mouse model was used to evaluate CETP expression in adipose, kidney, and retinal tissues of 32-week-old mice . Westernp = 1.00 × 10−6), whereas A-alleles of rs4783961 and rs1800775 were associated with HDL-C increases of 1.71 mg/dL and 0.91 mg/dL per A-allele, respectively, at nominal significance level , in the non-diabetic controls. We also observed that at a nominal significance level, the A-allele of rs4783961 was associated with a decreased risk of T2D  and the A-allele of rs1800775 was associated a reduced risk of DKD . However, these results were not adjusted for multiple testing, and thus should be confirmed in other larger, independent studies. HDL-C may stimulate pancreatic β-cell insulin secretion, modulate glucose uptake in skeletal muscle, and contribute to the pathophysiology of T2D [In the present study, three SNPs located in the promoter region were observed to be independent of age and sex and were significantly associated with HDL-C levels. In particular, SNP rs3764261 was associated with an HDL-C increase of 2.86 mg/dL per A-allele (y of T2D . In addiy of T2D . Furthery of T2D . Neverthy of T2D .1C. We believe that the difference in BMI between T2D patients and non-diabetic controls was eliminated from the analysis models. Third, the gene–diet interaction between CETP genetic polymorphisms and dietary fat intake could have an effect on plasma HDL-C level [There are several limitations to the present study. First, we did not measure CETP activity, and thus it is unclear whether the CETP SNPs affect CETP activity and whether they are associated with the risk of T2D or DKD. CETP promoter SNPs rs3764261 and rs4783961 were also investigated previously . The aut-C level , and becCETP expression in the T2D mouse model was decreased in the kidney and retinal tissues compared with that in control mice but was significantly decreased only in kidney tissue. We previously reported that db/db T2D mice exhibited features of the early clinical stages of DR at 32 weeks of age . Here, wIn conclusion, CETP variants in the promoter region may affect HDL-C levels. Taiwanese individuals possessing an allele associated with higher HDL-C may have lower risks of T2D and DKD.


Apolygus lucorum Meyer-Dür, has been an important pest of cotton crop in China, and is primarily controlled with insecticides, such as pyrethroids. To elucidate the potential resistant mechanisms of A. lucorum to lambda-cyhalothrin, a series of biological, biochemical, and molecular assays were conducted in the reference (AL-S) and lambda-cyhalothrin-resistant (AL-R) populations. Comparison of the molecular target of pyrethroid insecticides, voltage-gated sodium channel, revealed that there were no mutation sites in the resistant population, indicating target insensitivity is not responsible for increased resistance of AL-R to lambda-cyhalothrin. Furthermore, the synergism assays and the activities of detoxification enzymes were performed to determine detoxification mechanism conferring the lambda-cyhalothrin resistance. In the tested synergists, the piperonyl butoxide had the highest synergism ratio against lambda-cyhalothrin, which was up to five-fold in both populations. In addition, the result also showed that only cytochrome P450 had significantly higher O-deethylase activity with 7-ethoxycoumarin (1.78-fold) in AL-R population compared with AL-S population. Seven cytochrome P450 genes were found to be significantly overexpressed in the resistant AL-R population compared with AL-S population. Taken together, these results demonstrate that multiple over-transcribed cytochrome P450 genes would be involved in the development of lambda-cyhalothrin resistance in AL-R population.The mirid bug,  Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) had been a primary pest of cotton in northern China during the 1950’s, but its population densities always remained low owing to the frequent application of synthetic insecticides against Lepidopteran pests GX[E/D]T[T/S]), the helix K motif (EXXRXXP), the heme-binding “signature” motif (PFXXGXXXCXG) and a sequence motif (PXXFXP) specific to CYP6 members. The results indicated that these CYPs belong to typical microsomal P450 clades. The phylogenetic tree, generated from aligned amino acid sequences of CYPs, revealed that these CYPs were closely related to those of families CYP4 and CYP6 of other invertebrate species  and pyrethroid resistance [Lygus species found that resistance was correlated with increased activity of P450 detoxifying enzymes [Pyrethroid resistance mechanisms were usually complex, which mainly based on the pest, field environment, and insecticide application. A previous study of pyrethroid resistance in itution L15F and p enzymes . In the A. lucorum [P. vulgaris containing pesticide residue. The further biochemical assays confirmed indeed that the resistant individuals had higher level of P450 activities compared with reference individuals. These evidences pointed to a P450-mediated metabolic resistance mechanism involved in lambda-cyhalothrin resistance of AL-R population. Nevertheless, other metabolic mechanisms, such as glutathione S-transferase, and esterase mediated metabolisms, should not be excluded, because synergists DEM, DEF, and TPP also increased the toxicity of lambda-cyhalothrin in AL-R population. This phenomenon was similar with the enhanced detoxification rather than target insensitivity mechanism found in deltamethrin resistant L. striatellus [Xu et al found that the synergism of PBO to lambda-cyhalothrin was obvious with the synergism ratio up to 7.2 compared with the other three types of insecticides in  lucorum , which wiatellus .CYP6HM1, CYP6HM2, CYP6JB1, CYP6JB2, CYP6JC1, CYP6X2, and CYP395H1 are more highly increased in the AL-R population than the AL-S population (L. lineolaris was associated with pyrethroid resistance [L. lineolaris (up to 82% amino acid sequence identity). CYP6AY3v2 in Laodelphax striatellus  associated with deltamethrin resistance [L. striatellus. CYP6F1 in C. quinquefasciatus resistant to pyrethroids [Ceratitis capitate was resistant to lambda-cyhalothrin [CYP6X2 was also induced in both the AL-R (1.54-fold) and AL-S populations (1.86-fold) when adults were treated with a dose of lambda-cyhalothrin equivalent to their corresponding LD50 values. Therefore, we hypothesize that CYP6X2 gene may play a relevant role in the resistance of the AL-R population to lambda-cyhalothrin by over-expression of a lambda-cyhalothrin-inducible gene. However, the over-expression of P450 genes does not necessarily correlate with insecticide resistance [In the Order Hemiptera, a variety of studies have documented pyrethroid resistance associated with P450s , 33, 34,pulation . All of sistance , while oethroids . CYP6A51alothrin . Moreovealothrin –46. In osistance . FurtherCYP6D1 gene in permethrin-resistant strains of M. domestica, which was absent in susceptible strains [CYP6X2.Elevated expression of P450 genes in resistant insects may be achieved through increased transcription by mutations/insertions/deletions in cis-acting promoter sequences . There h strains . TherefoThe metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant AL-R population. Although, our data could not firmly conclude that up-regulation of the seven identified detoxification genes are associated with the observed lambda-cyhalothrin resistance, it certainly provides a solid basis for future functional studies of encoded proteins and resistance mechanism confirmation.S1 FigConserved amino acid domains common to cytochrome P450s are highlighted as follows: the helix I, helix K, PERF and heme-binding motif is shaded in yellow, blue, grey and purple respectively. A CYP6X2 B CYP6JB1 C CYP6HM1 D CYP6JC1 E CYP6HM2 F CYP6JB2 G CYP395H1.(RAR)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.