Cytochrome P450 monooxygenases (P450s) represent the largest enzyme family of the plant metabolism. Plants typically devote about 1% of the protein-coding genes for the P450s to execute primary metabolism and also to perform species-specific specialized functions including metabolism of the triterpenes, isoprene-derived 30-carbon compounds. Triterpenes constitute a large and structurally diverse class of natural products with various industrial and pharmaceutical applications. P450-catalyzed structural modification is crucial for the diversification and functionalization of the triterpene scaffolds. In recent times, a remarkable progress has been made in understanding the function of the P450s in plant triterpene metabolism. So far, ∼80 P450s are assigned biochemical functions related to the plant triterpene metabolism. The members of the subfamilies CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A are generally conserved across the plant kingdom to take part in plant primary metabolism related to the biosynthesis of essential sterols and steroid hormones. However, the members of the subfamilies CYP51H, CYP71A,D, CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y are required for the metabolism of the specialized triterpenes that might perform species-specific functions including chemical defense toward specialized pathogens. Moreover, a recent advancement in high-throughput sequencing of the transcriptomes and genomes has resulted in identification of a large number of candidate P450s from diverse plant species. Assigning biochemical functions to these P450s will be of interest to extend our knowledge on triterpene metabolism in diverse plant species and also for the sustainable production of valuable phytochemicals. Triterpenes, 30-carbon compounds originated from the 5-carbon isoprene units, constitute a large and structurally diverse class of natural products . The maj2,3-Oxidosqualene is a common biosynthetic precursor for the diverse triterpene skeletons produced in plants. Oxidosqualene cyclases (OSCs) catalyze the first diversifying step for triterpene biosynthesis by converting 2,3-oxidosqualene to a variety of cyclic triterpene scaffolds . In majoTo date, >23,000 triterpene structures are known from the natural sources. These are build-up on >100 structural scaffolds ranging from acyclic to hexacyclic structures . Among tTriterpenes are generally produced from the acyclic 30-carbon precursors squalene in bacteria and 2,3-oxidosqualene in eukaryotes. However, a few exceptions were also known. Green algae produce botryococcene and their methylated derivatives from farnesyl pyrophosphate . MoreoveThe conversion of 2,3-oxidosqualene into diverse cyclic triterpenes by OSCs, the class II terpene synthases is the first diversifying step of the triterpene biosynthetic pathway, and also marks the branch point for the biosynthesis of the sterols and steroid hormones . Unlike Figure 1). Besides the clans CYP51 (member: CYP51G) and CYP85 , the clans CYP72 (member: CYP734A), CYP86 (member: CYP94N) and CYP710 (member: CYP710A) were known for the sterol scaffold modifications .P450s representing about 1% of the protein coding genes of the plant genomes, constitute the largest family of the enzymes involved in plant metabolism . P450s aPhytosterols , steroid hormones , steroidal alkaloids , and steroidal saponins  are all derived from the cycloartenol scaffold. P450s involved in modification of the sterol scaffolds are discussed in the following sections.Figure 2). CYP51 function remained conserved in fungi and animals as 14α-demethylase of sterol precursors such as lanosterol, dihydrolanosterol, and eburicol. In animals and fungi, CYP51 members are designated as CYP51A and ERG11/CYP51F, respectively. Moreover, protozoan and bacterial genomes also encoded sterol 14α-demethylase, designated as CYP51E and CYP51B, respectively. The bacterial genomes possibly gained CYP51 members through horizontal transfer from the eukaryotic genomes . Moreover, CYP710A2 also converted campesterol to crinosterol and 24-epi-campesterol to brassicasterol . However, based on gene over-expression studies in Arabidopsis, the in planta functions of CYP710A1 and CYP710A4 in stigmasterol biosynthesis, and CYP710A2 in brassicasterol and crinosterol biosynthesis were established . The genetic screening of the mutant lines and biochemical analysis of the corresponding enzymes established the role of the CYP90B and CYP724B subfamily members in catalyzing the conversion of campesterol to 22-hydroxy-campesterol in Arabidopsis, rice, and tomato (The biosynthesis and catabolism of brassinosteroids (BRs), the poly-hydroxylated steroid hormones that mediate plant growth and development, involve P450s-catalyzed regio- and stereo-specific oxidation of the campesterol scaffold at the C2, C3, C6, C22, C23, and C26 positions. The P450 family members  that catalyze the consecutive biochemical reactions of the BRs pathway, were identified . These P450s were biochemically characterized by employing a protein expression platform based on Sf9 insect cells  was identified from the basal eudicot ynthesis . CYP716AFigure 1). P450 functions in mediating structural diversification of the pentacyclic triterpenes are discussed in the following sections.Oleanane, ursane, and lupane, derived from β-amyrin, α-amyrin, and lupeol, respectively, represent the major pentacyclic triterpene scaffolds . To dateCYP716A1 and CYP716A2 with PEN3, an OSC . A majority of these CYP716As catalyzed sequential three-step oxidation of the amyrin/lupeol scaffolds, leading to the consecutive formation of the hydroxyl, aldehyde, and carboxyl moieties at the C28 position . Tomato CYP716E26 and Centella asiatica CYP716E41 were biochemically characterized as C6β-hydroxylases that accepted α/β-amyrin and oleanolic acid/ursolic acid/maslinic acid as substrates, respectively , CYP51H10 (sad2) was found to be dispensable for essential sterols biosynthesis. However, CYP51H10 was indispensable for the production of anti-microbial oleanane-triterpene saponins (avenacins) that conferred disease resistance in oats toward the root-infecting fungal pathogens , suggesting the conserved role of the CYP93E in legume-specific triterpene saponin biosynthesis (Saccharomyces cerevisiae). The highest activity was obtained for the Phaseolus vulgaris CYP93E9 that showed 61-fold higher activity than the Medicago truncatula CYP93E2 (The members of the CYP93 family comprising of five subfamilies (A–E) were found in several plant genomes . The maj CYP93E1 . This enynthesis . Despite CYP93E2 . HoweverM. truncatula and Glycyrrhiza uralensis were biochemically characterized and were found to be associated with the legume-specific triterpene saponin biosynthesis. So far, four CYP72As of M. truncatula  and one CYP72A of G. uralensis (CYP72A154) were reported to partake in oleanane triterpene scaffold oxidation  to soyasapogenol B. However, CYP72A67 and CYP72A68 were found to be C2β-hydroxylase (oleanolic acid/hederagenin C2β-hydroxylase) and C23-oxidase (oleanolic acid/hederagenin/2β-hydroxy-hederagenin C23-oxidase) of the hemolytic saponin pathway, respectively  plants has various pharmaceutical properties and is being used worldwide as a natural sweetener (M. truncatula CYP72A63 was found to possess C30-oxidase activity and converted β-amyrin to 11-deoxoglycyrrhetinic acid following a three-step oxidation (Catharanthus roseus (CYP72A1 and CYP72A224) that participate in indole alkaloid biosynthesis . Among these, Lotus japonicus CYP71D353 had been shown to oxidize lupane-type pentacyclic triterpene scaffold . CYP71D353 still remains the only known member of the CYP71D subfamily with a role in triterpene biosynthesis. Some other CYP71D subfamily members were known to take part in monoterpene and flavonoid hydroxylation . To date, CYP87D16 of Maesa lanceolata is the only known member of the CYP87D subfamily associated with the pentacyclic triterpene scaffold modification . These are CYP716A47 (renamed as CYP716U1) and CYP716A53v2 (renamed as CYP716S1v2) of Panax ginseng, CYP87D18 of Siraitia grosvenorii, CYP81Q58 and CYP88L2 of Cucumis sativus, CYP87D20 of C. sativus, C. melo, and Citrullus lanatus and CYP81Q59 of C. melo and C. lanatus . Arabidopsis CYP71A16 was found to hydroxylate both marnerol and marneral at the C23 position, when expressed in yeast along with an OSC, marneral synthase (MRN1)  and over-expression lines . These genes co-expressed and clustered in the Arabidopsis genome with the OSC thalianol synthase  and tricyclic  triterpenes metabolism (e (MRN1) . Moreoveon lines . CYP71A1on lines . CYP708Asynthase .Figure 5). This triterpene degradation pathway was found to contribute to the formation of the pathogen-induced volatile terpenes in Arabidopsis  mutants that either could not produce saponins in root or had reduced level  with a C28 carboxylation  could be detected in iha mutants, similar to the wild type counterpart  that lacked a C2 hydroxylation could be detected in CYP72A67 mutants  that resulted in silencing of the CYP716A14v2 transcript expression  and sweet basil (Ocimum basilicum) P450s of the tetracyclic and pentacyclic triterpene pathways, respectively, was determined following transient gene silencing approaches. The in planta role of CYP88L2 and CYP81Q58 in cucurbitacin biosynthesis was probed by silencing their transcript expression in the cotyledons using a transient RNAi system. The down-regulation of CYP88L2 and CYP81Q58 transcripts resulted in decreased level of cucurbitacin in the cotyledons, confirming their involvement in cucurbitacin biosynthesis  was clarified following a virus induced gene silencing (VIGS) approach  of the Arabidopsis thalianol gene cluster was precisely determined following biochemical analysis of the RNAi or T-DNA insertion lines . CYP708A2 mutant line lacked thalian-diol  and, however, accumulated increased level of thalianol . CYP705A1 and arabidiol synthase clustered in the Arabidopsis genome. These genes co-expressed in roots, and also responded to the jasmonic acid treatment and pathogen infection -4,8-dimethyl-1,3,7-nonatriene (DMNT) production in roots has been well documented . CYP705Anfection . The genCYP71A16 T-DNA mutant lines accumulated marnerol, an alcohol spontaneously produced from marneral. Moreover, hydroxylated marnerol derivatives were detected in CYP71A16 and MRN1 over-expression lines  was also determined following biochemical analysis of the T-DNA mutant and over-expression lines . Unlike on lines . These relopment .1; Medicinal Plant Genomics Resource 2), an increasing number of P450s are expected to be assigned function in the plant triterpene metabolism. The future challenges are functional characterization of the P450s having unusual biochemical properties, and utilization of the P450s for the plant improvement program and for the production of valuable phytochemicals.So far, about eighty P450s were assigned specific functions to the plant triterpene metabolism . Considering a huge structural diversity of the plant triterpenes, it is quite obvious that several other P450s with uncharacteristic biochemical features are yet to be known. With the availability of the large scale genomic and transcriptomic sequence information (The 1000 plants S. cerevisiae), the in planta function of a limited number of these P450s was determined. Therefore, extending the functional genomics tools  to the non-model plants is crucial to know the biochemical function of the species-specific P450s and also to establish the biological roles of the triterpene specialized metabolites in plants.Nevertheless, notable advances in understanding the roles of the P450s in plant triterpene metabolism for the general as well as species-specific functions have been made. Usually CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A subfamily members are found to metabolize sterols and steroid hormones that mediate primary functions in plants. However, CYP51H, CYP71A,D CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y subfamily members are generally found to metabolize triterpenes that showed species-specific distribution in plants. The physiological roles of the P450s in metabolism of the sterols and steroid hormones were mostly revealed based on gene function analysis in the model plants like Arabidopsis, tomato, and rice. Although, a number of P450s were identified from non-model plants and biochemically characterized following protein expression in heterologous hosts, including model plants (Arabidopsis and tobacco) and microbe (SG collected literatures and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.05), and NeuN and CGRP mRNA and protein expression was markedly decreased (P < 0.05), but protein levels of Nestin, cleaved caspase 3, TNF-α, IL-1β and the ratio of Bax/Bcl-2 in SCI group were significantly increased (P < 0.05). The EA treatment significantly reduced miR-449a level and cleaved caspase 3, TNF-α, IL-1β level and the ratio of Bax/Bcl-2 (P < 0.01), but substantially increased Nestin, NeuN and CGRP expression (P < 0.05 or 0.01). High level of miR-449a in miR subgroup was accompanied by decreased expression of Nestin, NeuN and CGRP and increased expression of cleaved caspase 3, TNF-α, IL-1β and elevation of the ratio of Bax/Bcl-2 (P < 0.05), suggesting miR-449a inhibits the effects of EA on NSCs and neurons. Luciferase reporter assay showed that miR-449a bound to the 3' UTR of CGRP, and thereby regulated CGRP expression. In conclusion, EA promotes proliferation of neural stem cells and the survival of neurons by downregulation of miR-449a expression.The aim of this study is to investigate the mechanism of electro-acupuncture (EA) on the recovery of injured spinal cord. Rats were randomly divided into normal control, sham-operated, SCI, SCI+EA group and T10 segment spinal cord injury (SCI) rat model was established by the modified Allen's method. After 7 days, the mRNA and protein expression of Nestin, neuron specific nuclear protein (NeuN) and calcitonin gene related peptide (CGRP) were detected by real time RT-PCR, Western blot and immunohistochemistry respectively. The protein expression of cleaved caspase 3, Bax, Bcl-2, TNF-α and IL-1β were also detected by Western blot. MicroRNA 449a (miR-449a) expression was also compared. Further, 12 SCI rats were randomly divided into EA and miR subgroups (EA + miR-449a agomir injection). The expression of Nestin, NeuN, CGRP, cleaved caspase 3, Bax, Bcl-2, TNF-α, IL-1β and miR-449a was compared. The direct interaction of miR-449a and CGRP mRNA was assessed by dual luciferase reporter assay. At day 7, compared with sham-operated group, miR-449a expression in SCI group was significantly increased ( Spinal cord injury (SCI) is damage to the central nervous system (CNS) that causes temporary or permanent changes in muscle function, sensation, or autonomic function in parts of the body served by the spinal cord below the level of the lesion. To date, there is no effective treatment method for SCI primarily because most neurons in the adult CNS are terminally differentiated and cannot regenerate after injuries. A small number of neural stem cells (NSCs) have been recently identified in some regions of mature CNS such as the spinal cord. Therefore, the post-injury repair and regeneration of neurons and NSCs is crucial for the treatment of SCI. Currently, researches on SCI are mainly focusing on the regulation of genes that can stimulate the proliferation of neurons and NSCs. Nestin is a NSC-specific type IV intermediate filament protein that is required for the proper self-renewal of neural stem cells, and is closely associated with NSCs . NeuronAcupuncture is a traditional Chinese medicine treatment developed over 2500 years ago, and has been applied in the treatment of several neurological diseases . As an microRNAs are a class of small non-coding RNA molecules that are involved in a wide range of biological processes such as cell cycle, apoptosis, organ development, tissue regeneration, aging, and even the pathogenesis of several diseases through inhibition of target gene expression by binding to the 3'UTR region of these genes . Reverse transcription kit, and DreamTaq Green PCR Master Mix (2×) were purchased from Takara (Japan). Primers used in PCR amplification and miR-449a agomir (pre-miR-449a: 5'-CUGUGUGCGAUGGGUUGGCAGUGUAUUGUUAGCUGGUUGAGUAUGUAAAAGGCACCAGCUAACAUGCAACUGCUCUCCUAUUGCACAUACA-3') and the non-targeting sequence (negative control) were synthesized by GenePharma Biotech. . Protein extraction kit and BCA kit was purchased from Beyotime Institute of Biotechnology . Mouse anti-rat Nestin monoclonal antibody , mouse anti-rat NeuN and CGRP monoclonal antibodies , cleaved caspase 3 antibody, Bax antibody, Bcl-2 antibody, TNF-α antibody and IL-1β antibody , mouse anti-rat GAPDH monoclonal antibody  and HRP-labeled rabbit anti-mouse IgGand goat anti-rabbit IgG  were used in Western blot analyses. Envision detection systems  used in immunohistochemistry (IHC) was purchased from Dako . Electro-acupuncture therapeutic meter (G6805-1) was manufactured by Shanghai Medical Instruments Inc. . RPMI-1640 culture medium, fetal bovine serum (FBS), penicillin-streptomycin double-resistance and trypsin were purchased from Sigma . Quick-change site-directed mutagenesis kit was purchased from Stratagene Corporation . miR-449a mimic was synthesized by GenePharma Biotech. Firefly luciferase reporter vector pMIR-report luciferase was purchased from Ambion, Inc. . Lipofectamine 2000 transfection reagent was purchased from Shanghai Bioleaf Technology Co. . Opti-MEM reduced serum medium was purchased from Life Technologies, Inc. . Luciferase reporter gene detector GloMax20/20 was purchased from Promega Corporation . Gel imager GelDoc 2000 was purchased from Bio-Rad Corporation .Establishment of SCI model section. After the surgery, rats in the SCI+EA group were treated with an EA device at the Jizhong and Zhiyang acupoints  and raised at room temperature. After one week, 24 rats were randomly divided into 4 groups (n = 6 each): healthy control, sham surgery, SCI surgery, and SCI+EA group in order to evaluate the therapeutic effect of EA on SCI. Further, another 12 rats were subjected to SCI surgery and randomly divided into 2 subgroups: SCI+EA subgroup and miR group (SCI+EA+miR-449aagomir) in order to investigate the regulatory role of miR-449a. SCI surgery was performed as described below in The T10 segment SCI rat model was constructed by the modified Allen's method (weight-drop method). Briefly, rats were fastened for 12 h before surgery. Rats lying in the prone position were given an intraperitoneal injection of 300 mg/kg chloral hydrate. The skin around T10 segment in the back was disinfected and a longitudinal incision was cut. The erector spinae muscles were separated by blunt dissection, and the lamina and spinous process was removed to expose the T10 spinal dura. A small plastic round pad was placed on the exposed spinal cord. A 20-g object was vertically dropped through a 5 cm high glass tube onto the spinal cord. The muscle and skin was sutured. The SCI model was successfully constructed as indicated by the spasmodic swing of the tail, fluttering of the body and lower limbs, and flaccid paralysis of both lower limbs. -ΔCt method.At day 7, rats were anesthetized by an intraperitoneal injection of 300 mg/kg chloral hydrate. The T10 segment of the spinal cord was removed and homogenized on ice. Total RNA was extracted using the RNA extraction kit. RNA concentration was determined using a spectrometer under a wavelength of 260 nm. Total RNA was reverse transcribed into cDNA using the reverse transcription kit according to the manufacturer's instructions. The Nestin, NeuN, CGRP, and GAPDH mRNA, and miR-449a was measured by qRT-PCR using cDNA as template and GAPDH and small nuclear RNA U6 (snU6) as internal control. The reaction condition: 95 °C 15 s; followed by 30 cycles of 95 °C 5 s and 60 °C 60 s. After amplification, melting curve analysis was performed: 95 °C 15 s, 60 °C 30 s, 72 °C 30 s. The sequences of primers: Nestin, forward: 5'-CGCTCAGATCCTGGAAGGTG-3', reverse: 3'-TTGGGGTCCAGAAAGCCAAG-3'; NeuN, forward: 5'-GCAGATGAAGCAGCACAGAC-3', reverse: 5'-TGAACCGGAAGGGGATGTTG-3'; CGRP, forward: 5'-CTTGCTCCTGTACCAGGCAT-3', reverse: 5'-CACACCGCTTAGATCTGGGG-3'; GAPDH, forward: 5'-CTCCTGTTCGACAGTCAGCC-3', reverse: 5'-TGGAATTTGCCATGGGTGGA-3'; miR-449a, primer for RT:5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACaccagc-3', forward: 5'-GGGTGGCAGTGTATTGTTA-3', reverse: 5'-CAGTGCGTGTCGTGGAGT-3'; U6, forward: 5'-GCTTCGGCAGCACATATACTAAAAT-3', reverse: 5'-CGCTTCACGAATTTGCGTGTCAT-3'. The experiment was repeated three times. Data was analyzed using the 2The expression of Nestin, NeuN and CGRP in all groups and the protein expression of cleaved caspase 3, Bax, Bcl2, TNF-α and IL-1β in SCI+EA subgroup and miR group was compared by Western blot. Briefly, the T10 spinal cord of each rat was obtained and homogenized on ice. Total protein was extracted using protein extraction kits and quantified using a BCA kit according to the manufacture's instruction. Equal amounts of total protein (30 μg) were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was blocked in TBS buffer containing 5 % skim milk and 0.1 % Tween20 at room temperature for 2 h, and incubated with the appropriate primary antibody (1:1000) overnight at 4 °C. The membrane was washed with TBST and incubated with HRP-labeled rabbit anti-rat secondary antibodies (1:5000) at 37 °C for 2 h. The membranes were washed thoroughly with TBST for 5 min each and subjected to ECL detection. The intensity of bands was detected by a gel imager. The gray value of bands was analyzed by Image Lab 2.0 software (Bio-Rad Laboratories).2O2 for 10 min to block the activity of endogenous peroxidase followed by hot antigen repair. These sections were incubated in serum from nonimmune animals (rabbit or mouse) for 20 min at 37 °C before incubated with primary antibodies at 4 °C for overnight. Then sections were incubated with EnVision reagent (HRP-R/M) at 37 °C for 30 min and visualized with DAB reagent. Finally, sections were counterstained with hematoxylin and counted the positive cells by microscope.The protein quantity and distribution of Nestin, NeuN and CGRP was detected using immunohistochemical staining with EnVision detection systems. Briefly, paraffin sections were dewaxed and hydrated with tradition methods, then were incubated with 0.3 % H2. Cells in the logarithmic growth phase were used for subsequent luciferase reporter assay.Human embryonic kidney cells (HEK) 293T cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . Cells were cultured in RPMI-1640 medium containing 10 % FBS, 50 U/mL penicillin and 50 μg/mL streptomycin at 37 °C in an incubator with 5 % COEscherichia coli. Clones were selected and sequenced to obtain the CGRP-UTR fragment. Mutagenesis was performed at the miR-449a binding site using the Quick-change site-directed mutagenesis kit. The sequence of the mutated fragment CGRP-UTR-mut was confirmed by sequencing. The luciferase reporter plasmids pMIR-CGRP-UTR and pMIR-CGRP-UTR-mut were constructed using the firefly luciferase reporter vector pMIR-report luciferase. HEK 293T cells at 70 % confluence were respectively transfected with 500 ng of pMIR-CGRP-UTR, pMIR-CGRP-UTR-mut, and blank control pMIR, and 10 nM of miR-449a mimics or negative control using Lipofectamine 2000 transfection kit and cultured in Opti-MEM reduced serum medium and the luciferase activity was measured using dual luciferase assay kit following the manufacturer's instructions. The relative luciferase activity (RLA) was calculated as the ratio of firefly luciferase activity (Ff) to renilla luciferase activity (Rn).The CGRP gene  was analyzed using the online tool (http://www.targetscan.org) to predict the 3'UTR sequence for miRNA-449a. The primers were designed using Primer Premier 5.0 software and synthesized by GenePharma Biotech. The sense and antisense primer was 5'-CGCGGATCC ACTCTGCTAAACCTCAAGGGG-3', and 5'-CCGGAATTC GTCACAACATTACCATGTGTCCC-3', which separately contained a BamHI restriction site and an EcoRI restriction site. Total RNA of HEK 293T cells was extracted using the trizol kit and reverse transcribed into cDNA using the reverse transcription kit according to the manufacturer's instructions. The target sequence was amplified using cDNA as template. The amplified fragment was isolated by gel electrophoresis, extracted, ligated to T vector, and transformed into P values smaller than 0.05 were considered statistically significant.All experiments were repeated three times, and data were expressed as mean ± standard deviation. Statistical analyses were performed using SPSS 11.0 . Differences among groups were analyzed by one-way ANOVA followed by Tukey's test. P < 0.05), whereas miR-449a expression was significantly reduced after EA treatment . While the expression of Nestin protein in SCI group was to some extent higher than that in sham-operated group. We found no significant difference in Nestin expression between the two groups. When compared with SCI group, the Nestin, NeuN and CGRP mRNA and protein expression in SCI+EA group was significantly increased (P < 0.05 or 0.01). The immunohistochemical results also showed that Nestin protein level was raised to some degree, but NeuN and CGRP protein levels were markedly declined in SCI group (P < 0.01 or P < 0.05), which were all raised in SCI+EA group (P < 0.05 or 0.01) . But EA treatment after SCI could attenuate the promotion (P < 0.05 or 0.01) . Moreover, the results of real time PCR, Western blot and immunohistochemical staining were also shown that the expression of Nestin, NeuN and CGRP mRNA and protein in miR group was significantly reduced compared with EA group (P < 0.05 or 0.01)  , but did not affect the RLA in pMIR-CGRP-UTR-mut group . Cleavein vivo miRNA activity. In the current study, SCI rats were treated by EA combined with intraperitoneal injections of miR-449a agomir. It was found that miR-449a agomir upregulated the miR-449a level in these rats, which inhibited the high expression of Nestin, NeuN and CGRP. The result suggested that miR-449a mediated the stimulative effect of EA on Nestin, NeuN and CGRP expression. High level of miR-449a promoted the mature of caspase-3 and expression of TNF-α and IL-1β and the ratio of Bax/Bcl-2 which were suppressed by EA in ventral horn of injured spinal cord of rats. Furthermore, the regulatory mechanism of miR-449a on CGRP gene expression was also investigated by luciferase reporter assay. It was found that miR-449a mimic specifically bound to the 3'UTR of CGRP mRNA, revealing that miR-449a directly regulated the CGRP gene expression on the post-transcription level. MiR-449a is a recently identified member of the conserved miR-449 family. Studies have shown that miR-449 a/b level in liver cancer, bladder cancer, prostate cancer and gastric cancer is markedly lower compared with normal tissues, and exogenous miR-449 a/b expression can inhibit cell proliferation, and induce cell cycle arrest, senescence and apoptosis  in development of Traditional Chinese Medicine in Shanghai (Grant No: ZY3-JSFC-1-1008).The authors declare that they have no conflict of interest.


The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1.Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection.In this study, short-hairpin RNA (shRNA) expression cassettes targeting Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in  Autographa californica multiple nucleopolyhedrovirus (AcMNPV) for human IFN-β expression was reported in 1983 [Bombyx mori nucleopolyhedrovirus (BmNPV) [Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) [Escherichia coli [Baculoviruses have been widely used as vectors for protein expression since the generation of a recombinant  (BmNPV)  and Spodhia coli  or co-trhia coli –7, for tpolh) encoding for the occlusion body (OB) matrix protein, using the polh locus for foreign gene insertion. In addition to polh, the virus chitinase (chiA) and cathepsin (v-cath) genes involved in the release of OB are also dispensable for the virus replication in cultured insect cells. It has been reported that removal of baculovirus chiA and v-cath can improve the stability of exogenous proteins in different insect cells including S. frugiperda (Sf), B. mori (Bm), and T. ni (Tn) High Five™ cells [p10, which is abundantly expressed very late during infection and may compete with polh at a transcriptional level, together with its flanking genes p26 and p74, can also be deleted for enhancement of recombinant protein expression [To improve the quantity and quality of recombinant proteins produced in BEVS, considerable efforts have been made to modify baculovirus vectors in the past 30 years , 9. In te™ cells –13. AcMNpression .polh and p10 are the most commonly used controlling elements for efficient expression of foreign genes in BEVS. Earlier promoters have been utilized for heterologous protein production, but result in reduced productivity [Modification of foreign gene expression cassettes is another approach to increase recombinant protein production. The strong baculovirus late gene promoters of uctivity . In a reuctivity . Other ructivity , SUMO [1uctivity  and KDELuctivity  can alsoCampoletis sonorensis ichnovirus can delay lysis of baculovirus-infected cells and result in an increase of protein yield [BEVS is a transient expression system, the death of baculovirus-infected insect cells terminates protein production which peaks around 3 days post infection. Methods to slow cell death would enhance the productivity of this technology. It has been reported that introducing of vankyrin from the insect virus in yield .p35, p49, iap (inhibitor of apoptosis), and apsup (apoptosis suppressor) [p35 can reduce more than 1000-fold [Apoptosis, characterized by a series of morphological and biochemical features that are associated with cell death, is an important host defense mechanism to limit and control viral infections. Baculovirus replication in insect cells triggers shutoff of host protein synthesis and induces apoptosis , which ipressor) –26. Thes000-fold , 27.The Apoptotic process involves the activation of a series of proteases called caspases. Blocking the activity of caspases by RNA interference have been used to improve protein production in mammalian cells , 29. In Spodoptera frugiperda caspase-1 (Sf-caspase-1) and engineered the cassettes into a baculovirus vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis, and examined the production of reporter proteins green fluorescent protein (GFP), Discosoma sp. red fluorescent protein (DsRed) and firefly luciferase, in comparison with BEVS without shRNA.In this study, we constructed shRNA expression cassettes targeting E. coli strain HS996 cells as previously described [Spodoptera frugiperda 9 (Sf9) cells (Invitrogen) were cultured in T25 Flasks or 6-well plates at 27 °C in SFX insect medium  containing 1% fetal bovine serum (HyClone).The bacmid BAC10:KO1629, which contains an AcMNPV genome, was maintained in CCGCGGAGGTCGGGCAGGAAGAGGGCCTATTT-3′, with underlined SacII site) and HU6pR . The amplified DNA fragment was digested with double restriction enzymes SacII and XbaI and inserted into pTriEx-GFP [To make the constructs expressing short hairpin RNAs (shRNA), human U6 promoter was amplified by PCR from the genomic DNA of human embryonic kidney 293 T cells (the Cell Bank of the Chinese Academy of Sciences), using primers HU6pF , three pairs of oligomers were synthesized  were knocked out from the bacmid BAC10:KO1629 by Red/ET-based recombination. Briefly, rpsL-amp cassette was amplified by PCR using primers ChiF_rpsL (5’-CGAGTTCCAACTTGGCGCGTAGACTGTTGTTTGGTAGCCCAAATCCGTGAGATGGCCTGGTGATGG-3′) and ChiR_AMP(5’-TGTTTATAGTTAACAATGTCGGCAGCGTCTATGGCCATAGGAATAGGGCCTTACCAATGCTTAATC-3′), which were comprised of a homologous arm for ChiA/V-Cath (underlined) and the rpsL-amp cassette. The amplified fragment was transformed into HS996 competent cells containing the Red®/ET® plasmid pSC101-BAD-gbaA and bacmid BAC10:KO1629, and the 1928 bp ChiA/V-Cath fragment was replaced by the rpsL-amp cassette [The cassette . The resCGAGTTCCAACTTGGCGCGTAGACTGTTGTTTGGTAGCCCAAATCCGTGAAGGTCGGGCAGGAAGAGG-3′) and ChiR (5’-TGTTTATAGTTAACAATGTCGGCAGCGTCTATGGCCATAGGAATAGGGCCGGCATGAACATGGTTAGCAG-3′). The amplified fragment was transformed into HS996 competent cells containing the Red®/ET® plasmid pSC101-BAD-gbaA and bacmid BacΔCC(rspLamp), so that the rpsL-amp cassette could then be removed and replaced by U6Si1, and this generated BacΔCCSi1 with the knock-in shRNA1 gene in the bacmid backbone  endpoint dilution assay.Recombinant baculoviruses were produced by homologous recombination between co-transfected pTriEx-1.1 (Novagen) based plasmid and linearized bacmid using FuGENE HD Transfection Reagent (Promega). Baculoviruses generated by homologous recombination were harvested at 96 h post transfection (hpi), passaged two times, and titrated by a 50% tissue culture infective dose (TCIDSf9 cells infected by recombinant viruses at 48 hpi. The RNA samples were then treated with RNase-Free DNaseI (Thermo), and 2 μg of total RNA was used for reverse transcription performed using the 5× All-In-One RT MasterMix (ABM) according to the manufacturer’s instruction. The Sf-caspase-1 gene fragments were amplified by PCR using the primers Sf-casp1F (5’-TGTCAAACACCTTTTATG-3′) and Sf-casp1R (5’-TATTATGACACATGGGCA-3′). β-actin gene was also amplified as a control using primers actin-F(5′-GGTGGTTGTTAGATGTA-3′)and actin-R (5’-GCTGGGAGTGTATTTC-3′). After 25–29 cycles of amplification, the PCR samples were analyzed by 1% agarose gel electrophoresis.To detect the transcription level of Sf-caspase-1, total RNAs were isolated with the TRIzol RNA extraction kit (Beijing CoWin Biotech) from Sf9 cells were infected with recombinant baculoviruses at multiplicities of infection (MOI) of 5, harvested at 96 hpi by centrifugation at 500 g for 1 min, and then resuspended in PBS. Propidium iodide (PI) was added to the cell suspension to a final concentration of 10 μg/ml. After incubation at room temperature for 5 min in the dark, cells were analyzed by flow cytometry using a CyFlow Cube 6 flow cytometer (Partec).Sf9 cells were infected by recombinant baculoviruses and incubated for 2–5 days at 27 °C, and then dislodged from the plates. The fluorescence of GFP and DsRed in the re-suspended cells was detected by flow cytometry [ytometry , and theytometry . FireflySf9 cell is still unknown and the human U6 promoter has been found to be able to drive shRNA expression in Tn insect cells as efficiently as Drosophila U6 promoter [Long dsRNAs have been shown to be able to be expressed and interfere with the transcription of caspase-1 in stably transfected Sf and Tn cells –33, and promoter , human USf9 cells to generate recombinant baculoviruses by homologous recombination. The transcript levels of Sf-caspase-1 in insect cells were determined by semi-quantitative RT-PCR at two days post-infection (dpi) of the recombinant baculoviruses. As shown in Fig. Sf9 cells (Fig. The plasmid constructs carrying the shRNA expression cassettes were co-transfected with AcMNPV bacmid into lls Fig. . These rChiA/V-Cath could enhance protein production for both secreted and cytoplasmic proteins [ChiA/V-Cath was knocked out from AcMNPV bacmid BAC10:KO1629 and used as the knock-in locus for the Sf-caspase-1 shRNA1, which inhibited Sf-caspase-1 expression best among the three shRNAs (Fig. It has been reported that deletion of proteins , 12. To NAs Fig.  and b, gSf9 cells to generate recombinant baculoviruses expressing GFP by homologous recombination. The GFP fluorescence of the baculovirus infected cells was measured by flow cytometry at 2, 3 and 4 dpi. The assay was repeated at least three times and the mean fluorescence intensity of the Sf-caspase-1 repressed cells were constantly shown to be stronger than the control baculovirus infected cells (typical data shown in Fig. The bacmid BacΔCCSi1 carrying the Sf-caspase-1 shRNA1 expression cassette and control bacmid BacΔCC were respectively co-transfected with pTriEx-GFP into It has been proved that exogenous protein production in BEVS could be improved in caspase-1 suppressed stable insect cells –33. HoweTo further investigate the effect of Sf-caspase-1 suppression on fluorescent protein production, a red fluorescent protein DsRed, which is a tetramer and represents a protein where the maturation of the fluorophore is slow , was expTo investigate different kinds of recombinant protein production in the Sf-caspase-1 repressed BEVS, firefly luciferase, an oxidative enzyme that can produce bioluminescence, was expressed as a reporter. This enzyme requires the participation of chaperone components to achieve its correct folding to generate an active enzyme . By WestPrevious studies demonstrated enhanced protein production in caspase-1 suppressed cell lines even after 4 dpi , 31, 33.BEVS has been used in many labs as a routine implementation for recombinant protein production. In structural and biophysics studies, it has become the leading platform in producing diffraction-quality proteins among eukaryotic expression systems in recent years . The bacSf9 cells, and improved the activity of the expressed exogenous proteins in comparison with the BEVS not expressing shRNA. This baculovirus vector would be useful for the production of various recombinant proteins in insect cells, especially for the expression of active proteins for functional and structural studies or therapeutic applications.In the present study, we generated an AcMNPV shuttle vector carrying shRNA expression cassettes to silence Sf-caspase-1 expression. Infection with baculoviruses made from this vector suppressed the apoptosis of infected


The influence of porogens, swelling temperatures and crosslinking agents on the porosity of porous microparticles was carefully investigated. Porous P(S-DVB) microparticles were used as a packing material for high performance liquid chromatography (HPLC). Several benzene analogues were effectively separated in a stainless-steel column as short as 75 mm due to the high specific surface area of the porous microparticles. Porous P(S-DVB) microparticles were further sulfonated and subsequently modified with diazoresin (DR) via electrostatic self-assembly and UV (ultraviolet) radiation. After treatment with UV light, the ionic bonding between sulfonated P(S-DVB) and DR was converted into covalent bonding through a unique photochemistry reaction of DR. Depending on the chemical structure of DR and mobile phase composition, the DR-modified P(S-DVB) stationary phase performed different separation mechanisms, including reversed phase (RP) and hydrophilic interactions. Therefore, baseline separations of benzene analogues and organic acids were achieved by using the DR-modified P(S-DVB) particles as packing materials in HPLC. According to the π–π interactional difference between carbon rings of fullerenes and benzene rings of DR, C In the last few decades, a lot of investigation has been done on the synthesis and properties of porous P(S-DVB) beads. The activated swelling method, developed by Ugelstad et al. was utilized for the synthesis of monodisperse porous particles in the range of 1–20 μm . RecentlDue to lower back pressure, a more regular flow regime in the column and higher resolutions, which result from their excellent permeability and high surface area, the monodisperse microspheres with porous structures have been widely used in chromatographic columns as packing materials ,25,26,27In this paper, we synthesized monodisperse porous P(S-DVB) microspheres by utilizing the activated swelling technique and investigated the effect of the porogen ratio, temperature and crosslinking agents. The porous P(S-DVB) microparticles were further sulfonated with concentrated sulfuric acid and subsequently modified with diazoresin(DR)via electrostatic self-assembly and UV (ultraviolet) radiation. These porous microspheres were applied as column packing material in mix-mode HPLC analysis of benzene analogs, organic acids and fullerenes.As illustrated in The porosity of porous polymer microparticles is greatly influenced by porogens. The swelling temperature can also influence the morphology of porous P(S-DVB) microparticles. As shown in Using different crosslinking agents for synthesizing porous P(S-DVB) microparticles presents different morphologies. 2 sorption isotherms by utilizing the BET method -4 microparticles calculated by using the BET method. The average pore size of the P(S-DVB)-3 and P(S-EDMA) are 10 nm and 18 nm, respectively. The hysteresis loops at moderate to high relative pressure, as shown in The surface areas are calculated from NT method . The spe2 upon UV irradiation, then a SN 1 type of nuclear displacement by sulfonate occurs [2/g) and the distribution of pore size are shown in DR-modified porous P(S-DVB) microparticles were synthesized by following the routes in e occurs . The spe−1 indicate the successful sulfonation of porous P(S-DVB) microparticles. The absorption peak at 1265 cm−1 can be assigned to the stretching vibration of N-H derived from DR; the peaks at 1450 cm−1 and 1500 cm−1 were assigned to the stretching vibration of the aromatic ring of DR, demonstrating the presence of DR after the modification.As shown in v/v = 7:3). The back pressure of the column packed with P(S-DVB)-4 is lower than that of P(S-DVB)-3, because the average pore size of P(S-DVB)-4 (~18 nm) is bigger than that of P(S-DVB)-3 (~10 nm).To investigate their chromatographic performance, the porous P(S-DVB) microparticles were packed into stainless-steel chromatographic columns. The mixture of uracil, toluene, naphthalene and fluorene were separated by the packed column with various permeable polymer microparticles. As shown in Depending on the chemical structure of DR and mobile phase composition, the DR-modified porous P(S-DVB)-4 stationary phase performed different separation mechanisms, including reversed phase (RP) and hydrophilic interactions. As shown in n = 5) relative standard deviation(RSD) of retention time for the benzene analogues was less than 1%, day-to-day (n = 7) RSD was less than 2.5%, and column-to-column (n = 5) RSD was less than 3.5%. After a continuous 100 times running in one column, the RSDs of retention time for the benzene analogues were all less than 2.5%, and the separation performance of the P(S-DVB)-DR column was not deteriorated. Therefore, the P(S-DVB)-DR columns demonstrated very good stability and repeatability.With the introduction of hydrophilic secondary amine groups on the surface of the P(S-DVB)-4 particles, a series of organic acids were baseline separated on the P(S-DVB)-DR column b. The ban = 5) RSD of retention time for the organic acids was less than 1%, day-to-day (n = 7) RSD was less than 2%, and column-to-column (n = 5) RSD was less than 3.5%. After a continuous 100 times running in one column, the RSDs of retention time for the organic acids were all less than 2.5%, and the separation performance of the P(S-DVB)-DR column was not deteriorated. Thereby, the DR-modified P(S-DVB)-4 particles were robust and had excellent performance in mix-mode HPLC separations.60 and C70—a class of special nanomaterials—were also separated in the P(S-DVB)-DR column using hexane and isopropanol (v/v = 1:3.5) as eluent. Considering that both C60 and C70 have carbon ring structures, the π–π affinity interactions between benzene rings of P(S-DVB)-DR particles and the carbon rings of fullerenes make the separation practical. As shown in 60 and C70 were successfully separated on the P(S-DVB)-DR column (60—having less π–π bondings than C70—was first eluted due to the weaker interaction with the benzene rings of DR. Compared with According to the excellent performance of the P(S-DVB)-DR column in the separation of benzene analogues, CR column b, while R column a. Theref2, 99%) were purchased from Tianjin Chemical company. Poly(N-vinylpyrrolidone)  and polyvinyl alcohol  were provided by Sinopharm Chemical Reagent Company . Uracil (99%) and fluorene (99.5%) were purchased from Aladdin Chemical Reagent Company . Benzoyl peroxide  was bought from Tianjin Beichen Chemical Company . DVB and St were used after vacuum distillation. DR (Mn = 2500) was synthesized by polycondensation of diphenylamine-4-diazonium salt and paraformaldehyde in concentrated H2SO4 according to a method described elsewhere [Styrene , DVB (99%), AIBN (98%), sodium dodecylsulfate , DBP (99%), toluene (99.5%), naphthalene (99.5%), ethanol (99.5%), ACN (99.9%), isopropanol (99.7%) and THF (99%), copper chloride  were ultra-sonicated to be well dispersed in 10 mL distilled water. A mixture of 1.2 mL toluene and 1.2 mL DBP was emulsified in 20 mL 0.375 wt % SDS aqueous solution in an ultrasonic bath  separately. The dispersion of PS particles and emulsion of porogens were mixed and mechanically stirred at 300 rpm in an oil-bath at 35 °C for 24 h. A mixture of 0.6 mL styrene and 2.0 mL DVB containing 0.12 g BPO was emulsified in 30 mL 0.25% SDS aqueous solution using ultrasonic agitation . The prepared emulsion was added into the swelled PS dispersion solution and stirred for another 24 h. At the end, 3.5 mL of 10% PVA aqueous solution was added into the dispersion. At the polymerization stage, the dispersion was purged with nitrogen for 30 min. Polymerization of the monomer phase in the swollen seed particles was carried out at 70 °C under mechanical stirring of 120 rpm for 24 h. After polymerization, the particles were isolated, washed with ethanol three times by using a centrifugation–decantation protocol, and then extracted with THF at 60 °C for 12 h to remove linear polymer and swelling agent completely. After centrifugation, the porous microparticles were washed with ethanol and dried in a vacuum oven.2 for 15 min. Consequently, DR was covalently linked to the surface of porous P(S-DVB) microspheres [Modification of porous P(S-DVB) microspheres with DR was carried out as follows: the porous P(S-DVB) microparticles (0.5 g) were first sulfonated by concentrated sulfuric acid (20 mL) at 40 °C for 4 h. The sulfonated porous P(S-DVB) microspheres were then added into 10 mL aqueous solution of DR (4 mg/mL) under magnetic stirring. The modified porous microspheres were washed with deionized water three times by centrifugation, and this process was repeated another three times in order to ensure that DR was completely coated onto the porous microspheres. After being dried under vacuum at room temperature for 12 h, the DR-coated P(S-DVB) microspheres were exposed under 365 nm UV light with an intensity of 350 mW/cmThe surface morphology and structure of PS seed particles and porous P(S-DVB) spheres were observed by using scanning electron microscopy  and transmission electron microscopy . An accelerated surface area and porosimetry analyzer  was used for the BET tests. Chromatographic study was carried out using a HPLC  equipped with a UV detector.v/v = 1:1) and packed into stainless-steel columns  by applying a chromatographic column packing machine  under a pressure of 18 MPa. The column was connected to the HPLC framework and flushed with methanol at a flow rate of 0.8 mL/min for 2 h to equilibrate the column until a constant UV baseline was obtained. A mixture of uracil, toluene, naphthalene and fluorene was separated in the HPLC column packed with porous P(S-DVB)-4 and P(S-DVB)-DR microparticles at a flow rate of 0.5‒1.0 mL/min at room temperature applying a UV detecting wavelength of 254 nm with ACN–water mixture (v/v = 7:3) as eluent. The organic acids mixture of formic acid, acetic acid, lactic acid and benzoic acid was also separated on the above two kinds of column under a flow rate of 0.5 mL/min at room temperature with phosphate buffer (pH = 4) and methanol (v/v = 85:15) as mobile phase at a detecting wavelength of 210 nm. In addition, C60 and C70 were separated on the modified P(S-DVB)-DR column using hexane and isopropanol (v/v = 1:3.5) as the mobile phase at a flow rate of 0.5 mL/min at a detecting wavelength of 290 nm.The monodisperse porous PS microparticles were dispersed in isopropanol/methanol -DR particles. Compared with bare P(S-DVB) packing material, the DR surface modification contributed to the high efficiency of the P(S-DVB) stationary phase for mix-mode separation.In this paper, porous P(S-DVB) microparticles with high specific surface areas were successfully synthesized by the two-step activated swelling method. The effects of the porogens, swelling temperature and crosslinking agent were investigated in detail. The largest surface area is obtained when the toluene to DBP porogens ratio is 3:1, and porous P(S-DVB) microparticles have the roughest surface when a swelling temperature of 25 °C is applied. The obtained P(S-DVB) particles were modified by sulfonation and self-assembly positive charged photosensitive DR on the surface. After treatment with UV light, the ionic bonding between sulfonated P(S-DVB) and DR was converted into covalent bonding through a unique photochemistry reaction of DR. Depending on the chemical structure of DR and mobile phase composition, the P(S-DVB)-DR stationary phase performed different separation mechanisms, including reversed phase and hydrophilic interactions. Thus, a variety of baseline separations of benzene analogues and organic acids was achieved by using the P(S-DVB)-DR particles as packing materials in HPLC. According to the π–π interactional difference between carbon rings of fullerenes and benzene rings of P(S-DVB)-DR, C


Simplexvirus genus, for which both the free gD structure and the gD/nectin-1 complex structure were reported at high resolutions. The structural and functional features of other alphaherpesviral gDs, however, remain poorly characterized. In the current study, we systematically studied the characteristics of nectin-1 binding by the gD of a Varicellovirus genus member, the pseudorabies virus (PRV). We first showed that PRV infects host cells via both human and swine nectin-1, and that its gD exhibits similar binding affinities for nectin-1 of the two species. Furthermore, we demonstrated that removal of the PRV gD membrane-proximal residues could significantly increase its affinity for the receptor binding. The structures of PRV gD in the free and the nectin-1-bound states were then solved, revealing a similar overall 3D fold as well as a homologous nectin-1 binding mode to its HSV counterpart. However, several unique features were observed at the binding interface of PRV gD, enabling the viral ligand to utilize different gD residues (from those of HSV) for nectin-1 engagement. These observed binding characteristics were further verified by the mutagenesis study using the key-residue mutants of nectin-1. The structural and functional data obtained in this study, therefore, provide the basis of receptor recognition by PRV gD.An early and yet indispensable step in the alphaherpesvirus infection is the engagement of host receptors by the viral envelope glycoprotein D (gD). Of the thus-far identified gD receptors, nectin-1 is likely the most effective in terms of its wide usage by multiple alphaherpesviruses for cell entry. The molecular basis of nectin-1 recognition by the gD protein is therefore an interesting scientific question in the alphaherpesvirus field. Previous studies focused on the herpes simplex virus (HSV) of the  Alphaherpesvirinae subfamily.Both herpes simplex virus (HSV) and pseudorabies virus (PRV) recognize nectin-1 as the cellular receptor. They utilize the envelope glycoprotein D (gD) on the virion surface to interact with nectin-1, initiating the virus infection. Although the molecular basis of nectin-1 binding by HSV gD has been successfully elucidated with high resolution structures, the atomic features of PRV gD interacting with the same receptor remains uncharacterized. Here, we show that PRV gD exhibits nano-molar affinities for both human and swine nectin-1, and deletion of the membrane-proximal loop in the gD ectodomain dramatically increases its receptor-binding avidity. We further solved the free and the nectin-1-bound PRV gD structures. The free gD structure reveals a canonical fold of an IgV-like core wrapped by the N- and C-terminal extensions as observed in the HSV homologs. The N-terminus of PRV gD, however, is shorter than that of HSV gDs. The solved complex structure demonstrates that PRV gD exhibits a homologous receptor-binding mode to the HSV counterpart. Nevertheless, several unique features at the PRV gD binding interface suffice the viral ligand to engage nectin-1 with a series of residues differing from the HSV amino acids. These observations not only delineated the molecular basis of PRV engaging nectin-1 but also enriched our knowledge on the receptor-binding mechanism of the  Alpha-, Beta- and Gamma-herpesvirinae, in the Herpesviridae family [Alphaherpesvirinae subfamily, is reported to infect both farming  and pet (e. g. cats) animals [There are three major subfamilies, e family . The thre family , 3. For e family . The cape family . Pseudor animals . Herpes  animals .Alphaherpesviruses contain multiple surface glycoproteins (e. g. more than 11 in HSV) in the virion envelope . An earlNectin-1 is an immunoglobulin (Ig)-like cell adhesion molecule . It playIn addition to tethering virus particles onto cell surface and facilitating the viral spread, gD is also believed to play a key role in triggering the membrane fusion cascade, thereby leading to the entry of alphaherpesviruses –26. SeveVaricellovirus genus and HSV in the Simplexvirus genus, respectively [To further delineate the receptor recognition basis of alphaherpesviruses, we set out to investigate the detailed interactions between PRV gD and nectin-1. Despite the significant homology between PRV and HSV, the two viruses belong to different genera  were determined to be 191 nM with HU-nectin-1  and 301 nM with SW-nectin-1 , respectively. Therefore, PRV gD recognizes the human and swine receptors with essentially the same binding affinity. This result is consistent with our observation in the cell fusion assay which shows that nectin-1 of the two species mediates gD-dependent CHO-K1 fusion with similar efficiencies shown in the previous section. Furthermore, the determined Kd values are also in good accordance with a previous study reporting an affinity of approximate 130 nM for the PRV-gD/HU-nectin-1 interactions [To gain further insight into the PRV-gD/nectin-1 interaction, we set out to characterize their binding features using the real-time surface plasmon resonance (SRP) assays. Previous studies have shown that, by truncation of the C-terminal trans-membrane and cytoplasmic domains, the entire ectodomain of PRV gD could be yielded as a recombinant protein in the soluble form . Followikinetics . The equKds of gD284 to the human and swine receptors were 16.1 nM  and 18.4 nM , respectively  gD form , we furtectively . These vKon) [Kon) and the decrease of its dissociation rate (by approximate 2-fold in Koff).According to the previous studies, the observed affinity-difference between the long and short forms of HSV gD stem mainly from the changes in their kinetic association rates (up to 40-fold in Kon) . The enhRwork = 0.169 and Rfree = 0.192  which is aligned in parallel with the first half of strand C''. The latter extension, with residues A165-P250, is much more extended. It structurally folds into four α-helices  and one small β-strand (str3), covering about half of the central-core outer surface . In totaAs expected, the structure of PRV gD is quite similar to those of its HSV homologs. Superimposition of our structure with previously reported HSV gD structures revealed well-aligned core domain and terminal extensions . DespiteRwork of 0.238 and an Rfree of 0.266  and the exposed α1' helix of the core domain . These gOverall, the nectin-1 binding mode of PRV gD resembles its HSV counterparts. All the three viral ligands utilize the terminal extensions to contact the CC'C''FG sheet of the receptor IgV domain. A detailed superimposition between the current structure and the previously reported HSV-gD/nectin-1 complex structures, however, revealed an obvious difference in their steric position (relative to the receptor) for the bound gD proteins. With well-aligned nectin-1 molecules, a shift-distance of about 3.5 Å was observed between the PRV and HSV gDs . Further2 in PRV gD and 1170.9 Å2 in SW-nectin-1 were buried upon complex formation. We therefore scrutinized this binding interface to delineate the amino acid interaction details between the two binding entities. Along the nectin-1 CC'C''FG sheet, the major receptor-engagement components of gD include its N-loop, the α1' helix in the IgV-core, and helices α2', α3', α3 and the α3'/α3 loop in its C-terminal extension    and ther> 40 μM) . The funKon/Koff rates have also been observed in other virus-ligand/receptor binding pairs when their interface residues are mutated [In contrast to F129 with 45 vdw contacts for PRV-gD, the intermolecule binding contributed by nectin-1 N77 (29 contacts) and M85 (20 contacts) were only about half of that for F129 . Accordi mutated .Simplexvirus genus in the Alphaherpesvirinae subfamily. The structures of both the free HSV gD and its complex with nectin-1 are reported at high resolutions [Varicellovirus member of the alphaherpesviruses, the PRV. Despite of its low sequence identity to the HSV homologs (~ 22%), PRV gD reserves the canonical gD features, including an IgV-like core with a kinked C” strand and the surface-exposed N- and C-terminal extensions. We further solved the complex structure of PRV gD bound with SW-nectin-1, which revealed a similar nectin-1 binding mode as observed for HSV gD. Nevertheless, several unique features at the PRV-gD/nectin-1 binding interface  when bound to the receptor) suffice the PRV ligand to recognize nectin-1 using quite different gD residues from those of the HSV homologs. These structural observations therefore provide a systematic view on the receptor binding mechanism of a second alphaherpesvirus and yet the first in the Varicellovirus genus.Of the thus-far identified alphaherpesviral gD receptors, nectin-1 is likely the most effective in terms of its wide usage by different viruses. HSV-1, HSV-2, PRV and BHV-1 are all reported to utilize nectin-1 for cell entry , 30, 31.olutions , 22, 25.It is interesting that both in the free PRV gD structure and in the PRV-gD/nectin-1 complex structure, a large C-terminal region of the gD molecule are untraceable. We noted that it has been a long time (over four months) before the proteins (gD337 for the free structure and gD284 for the complex structure) were successfully crystallized. Taking into account that large disordered parts of a protein are likely perturbing crystal formation, it may indicate a possible proteolysis of both the gD337 and the gD284 C-terminal regions during crystallization, leading to similar proportion of the gD residues (P7-P250 in the free gD structure and P9-D241 in the complex structure) being successfully traced in the free gD and the nectin-1 bound structures. We therefore believe that the re-orientation of the gD N-loop before and after nectin-1 binding is more likely the result of receptor engagement than arising from the differences in the gD forms used for crystallization. A flexible N-loop which experiences rearrangement or is structurally stabilized upon receptor binding has also been observed for HSV gDs , 26.With an overall similar structure, PRV and HSV gDs all engage the nectin-1 CC'C''FG sheet for receptor recognition. Along the sheet, a total of 20 residues  were recognized by all the three viral ligands , falling in a good accordance with the prelocation of PFD in gD [Kon between PRV gD284 and gD337 is only about 5–8 folds. This argues against a prelocation of the membrane-proximal loop in PRV gD and may indicate a novel mechanism of the loop interfering with nectin-1 binding. These are interesting issues that are worth of studying in the future.In HSV, the membrane-proximal loop of the gD ectodomain is proposed as the PFD which would interact with gB/gH/gL to trigger membrane fusion . AccordiFD in gD . In contIn contrast to HSV, both previous studies and the functional data in this report demonstrated that PRV can not use HVEM as a cellular receptor , 35. It in vitro SPR data) might facilitate the development of a gD-based subunit vaccine in the future.The PRV infection can cause pseudorabies  in pigs and other animals . Althoug50/ml PRV in DMEM) at 37°C for 3 h, after which 2% FBS was added to the medium. Cells were then photographed under microscope 72 h post infection.CHO-K1 cells  were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fatal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. For the PRV infection experiment, cells were seeded in the 6 well plate with 70% confluence. After 24h, cells were transfected with pcDNA4.0-HU-nectin-1, pcDNA4.0-HU-HVEM or pcDNA4.0-SW-nectin-1 expression plasmids, separately. After 24 h, cells were incubated with PRV Bartha-K61 solution (100 TCIDE. coli BL21 (DE3) and the inclusion bodies were prepared in a buffer composed of 100 mM Tris-HCl, pH 9.0, 400 mM L-Arginine, 2 mM EDTA, 5 mM reduced glutathione and 1 mM oxidized glutathione. Then, the refolded proteins were subjected to gel-filtration on a Hiload 16/60 Superdex 200 column . By estimation of their MWs, the monomer peaks could be obtained. The resultant monomeric proteins were carefully collected, concentrated, and then applied for kinetic studies using surface plasmon resonance.DNAs encoding the ectodomains of the human and swine nectin-1 (amino acids 30–335) were obtained by PCR using the primer pairs of human-Nectin1-F (5'- CGCGGATCCGTCCCAGGTGGTCCAGGTGAAC -3') / human-Nectin1-R (5'-AAGGAAAAAAGCGGCCGCTTCTGTGATATTGACCTC-3') and porcine-Nectin1-F (5'-CGCGGATCCGACCCAGGTGGTCCAGGTGAACG-3') / porcine-Nectin1-R (5'-AAGGAAAAAAGCGGCCGCCTCTGTGATATTGACCTCCACC-3'), respectively. The amplified fragments were then individually cloned into the prokaryotic expression vector pET21b (Invitrogen) with the BamHI and NotI sites. The proteins were then prepared following the reported methods [The DNAs coding for PRV (Becker strain) gD ectodomain of residues 1–284 (gD284) and 1–337 (gD337) were amplified by PCR with the same forward primer of PRV-gD-F (5'-CCGGAATTCAGGACGTGGACGCCGTGCC-3') pairing with different reverse primers of PRV-gD284-R (5'-CCGCTCGAGTTAGTGGTGATGGTGGTGGTGCCGCGTCGCCGGCTCGGGCAG-3') and PRV-gD337-R (5'-CCGCTCGAGTTAGTGGTGATGGTGGTGGTGGCGGTGGCGCGAGACGCC-3'), respectively. The coding sequence of the swine nectin-1 IgV domain (amino acids 37–143) was amplified using primers pNectin-1-IgV-F (5'-CCGGAATTCAGGACTCCATGTATGGTTTCATCGGC-3') and pNectin-1-IgV-R (5'-CCGCTCGAGTTAGTGGTGATGGTGGTGGTGCATCACAGTGAGGTTGAGCT-3'). The resultant DNA fragments that contain a C-terminal 6×his coding sequence were then individually cloned, via EcoRI and XhoI sites, into a previously modified pFastBac1 vector, which has been engineered to incorporate a baculovirus gp67 signal sequence at the N-terminus –41. The The mutant proteins of human nectin-1 ectodomain were prepared as previously described . The binThe kinetic analysis was performed by SPR using the BIAcore 3000 system. Gradient concentrations of PRV gD284 and PRV gD337 were flowed at 30 μL/min over nectin-1 (WT or mutant) immobilized at about 1,200 response units, and tested for binding at 25°C. The running buffer is composed of 10 mM HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20.All the crystals were obtained, with the hang-drop vapor-diffusion method, by initial screening with the commercial Hampton Research kits and then by condition optimizations. Free PRV gD crystal was finally obtained by mixing 1 μl of the concentrated gD337 protein at 10 mg/ml with 1 μL reservoir solution consisting of 0.1 M ammonium acetate, 0.1 M bis-tris pH 5.5, 17% w/v polyethylene glycol 10,000. To obtain the crystal of the PRV gD/SW-nectin-1 IgV complex, two proteins (gD284 and SW-nectin-1 IgV) were separately purified by gel-filtration chromatography, mixed at 1:1 molar ratio, and incubated on ice for 2 h. The mixture was further purified by gel-filtration chromatography using a Superdex 200 column  and the complex peak was carefully collected and then the PRV gD/SW-nectin-1 IgV complex was concentrated to 5 mg/mL. Diffractable crystals were finally obtained by mixing 1 μL of the protein complex with 1 μL reservoir solution consisting of 0.2 M sodium chloride, 0.1 M sodium acetate pH 5.5, 20% w/v PEG 10,000, followed by incubation at 4°C for about 4 months.The diffraction datasets were collected at beamline BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF) using the synchrotron radiation at 100K. The cryoprotectant solution is composed of 15% V/V glycerol and 85% V/V reservoir solution. Data were then processed with HKL2000  for indeThe fusion mediated by HSV gB/gD/gH/gL and the receptors has been validated in various cell types , 29. In


This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near  and remote from  the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency . The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block.Cardiac potassium channels encoded by human ether-à-go-go–related gene ( This current contributes to ventricular action potential repolarization and effectively controls the duration of the QT interval in humans (–torsades de pointes (TdP) arrhythmia, which can lead to sudden cardiac death  available for drug interactions. Attenuation of channel inactivation in N558K  on wildtype (WT) hERG and a series of alanine replacement mutants involving hERG pore residues previously shown to be important for drug block. A recent study reported a significant reduction of drug block in a hERG F557L mutant with a suggestion that this residue on the S5 helix . Washout was attempted in five cells with partial recovery to 59.5 ± 4.1% of control amplitude. Fig. 2C shows the mean concentration-response relationship for IhERG tail inhibition by Cavalli-2; a fit to the data using 50) value of 35.6 nm , 30.9–41.1) with a Hill slope of 0.69 . Voltage dependence of inhibition was assessed using a similar, modified protocol with test depolarizations to voltages between −30 and +60 mV . In each of control and Cavalli-2, the IhERG activation relationship was derived from current-voltage plots of IhERG tails, and V0.5 and k values were derived from these using a fit to the data with V0.5 and k values were, respectively, −16.0 ± 3.5 mV and 5.4 ± 0.8; in 30 nm Cavalli-2, these values were −21.1 ± 2.9 mV and 5.0 ± 1.5 . Fractional inhibition of IhERG was voltage-dependent over the tested range . IhERG tail inhibition was steeply dependent on voltage at membrane potentials coinciding with the rising phase of the IhERG activation relationship. An increase in IhERG was seen at −30 mV in Cavalli-2, which correlated with the leftward shift in voltage-dependent activation. Notably, fractional inhibition also showed some increase over voltages at which the activation relationship had reached a plateau. This feature was used to probe block further using Woodhull analysis ) waveform was assessed using AP voltage clamp . Fig. 2F shows that 30 nm Cavalli-2 inhibited peak IhERG during the AP by 42.3 ± 6.7%, which was not significantly different from that with the standard protocol shown in Fig. 2B .Five concentrations of Cavalli-2 on ratory 1820, 32–34analysis ), yieldiIhERG inhibition on channel gating. This was investigated further using an envelope-of-tails protocol .The results shown in protocol , 37–40, IhERG tails on repolarization to −40 mV elicited by the standard protocol in Fig. 2B were used to assess effects of Cavalli-2 on IhERG deactivation. Values for t½ deactivation were calculated from the peak tail current to the end of the repolarizing pulse in control and after application of 30 nm Cavalli-2. No significant change in t½ deactivation was observed in the presence of Cavalli-2 .IhERG inhibitors is significantly dependent on inactivation gating  was used to determine voltage-dependent availability of IhERG in control conditions and in the presence of Cavalli-2. Fig. 4B shows mean plots of IhERG availability, fitted with V0.5 of IhERG inactivation in control was −54.2 ± 2.2 mV, and k was 22.1, whereas in 30 nm Cavalli-2, the V0.5 was −65.8 ± 1.1, and k was 19.9 ± 1.1. Thus, Cavalli-2 caused a modest but statistically significant leftward shift in voltage-dependent inactivation , consistent with stabilization of the inactivated state by the drug. IhERG currents elicited by the third step following a brief repolarizing step to −120 mV were used to determine the time course of inactivation. The inactivation τ . Thus, Cavalli-2 did not statistically significantly alter the time course of development of inactivation.Taken together, the data in n gating , 22, 41.IhERG was investigated using the three-step protocol shown in Fig. 4D. Membrane potential was stepped from −80 to 0 mV for 3 s after which a further 4-s step to +40 mV to promote IhERG inactivation was applied before repolarization back to 0 mV for 3 s prior to returning membrane potential to −80 mV. The protocol was applied first in control solution and then following a 5-min exposure to Cavalli-2 at −80 mV in the absence of pulsing. In both conditions, outward current developed during the initial 0-mV command that was reduced during the +40-mV step, reflecting enhanced inactivation during that phase of the protocol. A supramaximal concentration (5 μm) of E-4031 was used to enable subtraction of background current at each phase of the protocol, and then fractional inhibition of IhERG was compared during the different phases of voltage command. Fig. 4E shows that fractional inhibition of IhERG by Cavalli-2 was significantly reduced during the +40-mV phase . This is consistent with a slightly stronger affinity of the compound for activated than inactivated channels.Inactivation dependence of the compound's effect on WT IhERG inactivation (IhERG (300 nm), shown in Fig. 5A, inhibited N588K IhERG by <50%  with an IC50 of 544 nm  and Hill coefficient (nH) of 0.97  and produced only a small effect on S620T IhERG  with an IC50 of 1916 nm  and nH of 0.63 . Thus, attenuation or removal of channel inactivation had very significant effects on Cavalli-2 action, consistent with an important role for inactivation in optimally exposing the binding site(s) on the channel for the compound.The role of inactivation gating was probed further using N588K and S620T attenuated inactivation mutants located in spatially distinct modules of the channel see . Asn-588tivation . A conceIhERG inhibitors have binding determinants within the hERG channel inner cavity that include residues at the base of the pore helix/selectivity filter and aromatic residues on the S6 helix  nm and nH of 0.99 . Fig. 6A compares the effects of 100 nm Cavalli-2 on S624A with its WT control, showing an attenuated effect for the mutant channel. Fig. 6B compares the effects of 100 nm Cavalli-2 on WT and T623A hERG at −120 mV, showing attenuated inhibition for the latter. Fig. 6C shows superimposed concentration-response relationships for WT and S624A IhERG; the IC50 for the mutant was 262 nm  with nH of 0.72 , 7-fold that of its WT control. Fig. 6D shows superimposed concentration-response relationships for WT and T623A IhERG; the derived IC50 for the latter was 281 nm  with nH of 0.54 , 16-fold that of its WT control.High-affinity methanesulfonanilide S6 helix , 15, 16.gh [K+]e –20, 33. A compares effects of 300 nm Cavalli-2 on Y652A IhERG (panel ii) with WT IhERG (panel i) under similar recording conditions. Although this concentration produced a profound inhibition of the WT channel, it had only a modest inhibitory effect on Y652A IhERG. Fig. 7B shows corresponding concentration-response relationships with an IC50 for Y652A IhERG of 594 nm  and nH of 0.74 , 17-fold that of its WT control. The Tyr-652 residue has previously been reported to influence voltage dependence of inhibition for some compounds , there was no significant voltage dependence to the inhibitory effect of the compound on Y652A IhERG tails . Fig. 7E shows the effect of 1 μm Cavalli-2 on F656A IhERG; this concentration reduced the current by ∼40%. Fig. 7F shows a concentration-response relationship for inhibition of F656A IhERG with a derived IC50 of 1315 nm  and nH of 0.93  superimposed with that for WT IhERG under similar conditions. This mutation had a profound effect on the inhibitory action of Cavalli-2: the derived IC50 was 75-fold that of its WT control.Fig. 7ompounds , 14, 42. channel D, there A compares the effect of 300 nm Cavalli-2 on WT (panel i) and F557L (panel ii) IhERG, showing a reduced effect of the compound on the mutant channel. Fig. 8B shows superimposed concentration-response relationships for WT and F557L IhERG. The derived IC50 for inhibition of F557L IhERG was 339 nm  with nH of 1.2 , 10-fold that of its WT control. The mechanisms by which Phe-557 influences drug binding to hERG are not yet well elucidated but in principle could involve direct or indirect effects; in some homology models, this residue lies close to Tyr-652 , there was no significant voltage dependence to the inhibitory effect of the compound on F557L IhERG tails . Thus, similar to Tyr-652, Phe-557 influences the voltage dependence of IhERG inhibition by Cavalli-2. Effects of all the mutations on IhERG-blocking potency of Cavalli-2 are summarized in A recent report has suggested a novel binding determinant for hERG-blocking drugs, residue Phe-557 located on the channel's S5 helix . The F55Fig. 8 Tyr-652 , althoug channel D, there 50 values of positively charged hERG blockers correlate with the number of noncovalent interactions between drug and pore residues in optimized hERG pore models .We searched for low-energy-score outputs for Cavalli-2 docked into the open pore structure of the recent cryo-EM structure  using GOe models . The sim0 values . In anal+ permeation pathway toward the S5 helix . The side chains of residues lining the pocket as well as the three Tyr-652 and Phe-656 residues nearest the selected pocket could rotate freely during docking to allow the drug to access the pocket and to optimize interactions with aromatic side chains, especially Phe-656. However, low-energy-score poses involving interactions with more than one Phe-656 side chain were not found.Wang and Mackinnon  highlighS5 helix  and 9. WA and Ref. e.g. Ref. i − 4 backbone carbonyl group (of Tyr-652) and, in the hERG structure, disruption of side chain packing at the interface of the S5 and S6 helices. Both GOLD and Flexidock reoriented the side chain back to the cryo-EM structure configuration during docking, allowing only a single Phe-656 interaction with Cavalli-2. Low-energy-score poses could be obtained with multiple (two) Phe-656 interactions by fixing the Cα–Cβ bond rotamer of Phe-656 residues adjacent to the pocket  to maintain the projection of these side chains toward the pore. A low-energy-score pose is shown in Figs. 9B and 11B. In this and similar low-energy-score poses, Cavalli-2 made aromatic π-π stacking interactions with two adjacent Phe-656 side chains and with Phe-557 and Phe-619 aromatic rings, but interactions with Tyr-652 were not favored.In the cryo-EM open pore structure, the Phe-656 side chains adopt an unexpected orientation, projecting away from the central pore (11C and 11C). In this pose, Cavalli-2 interacted with side chains of Phe-557, Phe-619, Thr-623, and Tyr-652 but only a single Phe-656 side chain (the side chain displaced into a pore-facing configuration).Interestingly, rotation of a single Phe-656 side chain toward the pore cavity could be obtained in unconstrained docking runs in configurations where one of the Cavalli-2 phenyl rings replaced the Phe-656 side chain in its location packed between the S6 and S5 helices C and 11C+ channel MthK because this model produces low-energy-score poses that are consistent with experimental mutagenesis data for hERG blockers . A representative low-energy-score pose is shown in +-binding site where the negative electrostatic potential from the C-terminal pore helix dipole charges is focused (blue star) so that all four functional groups of the drug made interactions with hERG. In these poses, Cavalli-2 could interact with Ser-624 (e.g. by direct or water-mediated hydrogen bonding with the protonated amino group) but not with the Phe-557 side chain. This type of pose for Cavalli-2 is consistent with a recent demonstration for several positively charged hERG inhibitors, using unnatural amino acid substitution, that neither Tyr-652 nor Phe-656 participate in cation-π interactions .Comparison with the MthK model is also useful in estimating a minimal rotation of the S6 helix in the hERG cryo-EM structure that would accommodate projection of the Phe-656 side chains toward the pore in a configuration compatible with multiple interactions with high-affinity hERG blockers. A small clockwise rotation of the S6 helix by 20–30° in the hERG structure would position the Cα carbon of Phe-656 in a position on S6 relative to the pore axis that is equivalent to that for the Phe-656 Cα atoms on S6 in the MthK model. Similarly, in a hERG model built onto the closed pore structure of the highly homologous rat EAG cryo-EM structure , a relatB and 11B). The best docking scores using both ChemPLP and Goldscore scoring (Table S1) were obtained with a hERG pore model built on the MthK template in which Cavalli-2 made multiple interactions with Phe-656 side chains (11D).In summary, interaction of Cavalli-2 with potential binding pockets of the hERG cryo-EM structure resulted in limited interactions with Phe-656 side chains that are expected from mutagenesis data  to dominFigs. 9e chains  and 11D.50 value for Cavalli-2 in human embryonic kidney (HEK) cells at 37 °C (36 nm) is similar to the value (17 nm) determined in HEK cells at room temperature by Cavalli et al.  suggests that channel activation is required for the drug to access its binding site. The δ value from the Woodhull analysis (0.2) is similar to that for other drugs that interact with residues within the hERG K+ permeation pathway . These features of Cavalli-2 block are compatible with those exhibited by drugs with more complex structures.Our ICi et al. , consiston curve D suggest pathway , 36, 44 IhERG nevertheless showed a clear reliance on the process of inactivation for maximal drug block as inhibition was significantly reduced for hERG mutants with attenuated inactivation. The reduction of drug block in these mutants is associated with attenuation of inactivation per se (rather than direct drug interactions with the mutated residues) because the attenuated inactivation N588K mutation resides in a helical structure in the hERG turret in the extracellular region of the hERG pore far from the drug-binding site  is not simply attributable to altered voltage-dependent activation of mutant compared with WT channels as the voltage dependence of activation of Y652A hERG has been shown to be similar to that of the WT channel . Phe-656 aromatic side chains are too far apart in this configuration to allow interaction of two phenyl rings on Cavalli-2 with two separate Phe-656 side chains while making interactions within a hydrophobic pocket. Only when Phe-656 side chain rotamers were constrained to reorient side chains toward the pore could multiple interactions between Cavalli-2 phenyl groups and multiple Phe-656 side chains be obtained (B). However, these rotamers are not favored due to Phe-656 side chain clashes with the Tyr-652 backbone carbonyl group and the loss of side chain packing between the S5 and S6 helices. If unconstrained during docking, the side chains reorient back to the configuration found in the EM structure. It is unlikely therefore that the apparent discrepancy between high dependence on Phe-656 and the limited interactions of Cavalli-2 with Phe-656 side chains in the cryo-EM structure can be resolved by postulating reorientation of Phe-656 side chain(s) during drug binding; more substantial conformational changes from the cryo-EM structure are required for Cavalli-2 to interact with more than one Phe-656 side chain as described below.Contrary to these expectations, docking of Cavalli-2 into the hERG cryo-EM structure with the binding site selected to promote interaction with one of the hydrophobic pockets fails to find low-energy-score poses in which Cavalli-2 can interact with more than one Phe-656 side chain. This results from the orientation of the side chains of Phe-656 that project away from the pore axis toward the S5 helix in the cryo-EM structure  and 11A.11obtained B. Howevei.e. loss of side chain packing between the S5 and S6 helices resulting from replacement of a bulky phenyl group with a methyl group in F656A results in a collapse of optimal configurations of drug-binding groups. It has been observed, for example, that retention of high binding affinity for MK-499 and cisapride can be retained if Phe-656 is replaced by bulky aromatic or aliphatic side chains, which should be more suitable than Ala for filling the space occupied by the Phe-656 side chain phenyl rings in the hERG EM structure , much get al.  and Phe-485 , cisapride sensitivity was conferred by shifting the aromatic amino acid one residue toward the C terminus, a movement equivalent to a clockwise rotation of the S6 helix by 100° (e.g. by 30–40°) would move the side chain of Phe-656 in the hERG EM structure to a pore-facing configuration that would allow interaction of Cavalli-2 with more than one Phe-656 side chain in a configuration similar to that for the MthK-based hERG pore model (S3). Because Cavalli-2 block shows approximately equivalent preference for an open and open-inactivated state , such a configuration resulting from reorientation of S6 is not the inactivated state per se but is only optimized in channels that are able to inactivate as described also by Chen et al. , including a requirement for inactivation competence in hERG channels and a strong dependence on Phe-656 for block. Docking using the new open pore hERG structure finds low-energy-score poses in which Cavalli-2 extends into hydrophobic pockets that were proposed to account for the unique promiscuity of hERG toward a variety of structurally diverse high-affinity blockers. However, in these states, Cavalli-2 does not make interactions with the multiple Phe-656 side chains that seem to be required for the strong dependence of Phe-656 on high-affinity block. The close structural similarity between the WT cryo-EM structure and a copublished inactivation-attenuated S631A hERG open pore structure, together with the evidence that high-affinity binding to inactivation-competent hERG channels may be associated with rotation of S6 helices to reorient Tyr-652 and especially Phe-656 aromatic side chains into optimal positions or high-affinity drug block  or Lipofectamine LTX (Invitrogen) following the manufacturer's instructions. The amount of hERG construct DNA that was transfected varied between 0.1 and 1.0 μg depending on the level of functional expression. 0.15 μg of CD8 was cotransfected as a transfection marker, and successfully transfected cells were identified using Dynabeads® (Invitrogen). Cells were plated on sterilized glass shards in 40-mm Petri dishes and incubated at 37 °C (5% CO2) at least 24 h before electrophysiological recording; this was to allow time for cell recovery and hERG construct expression.HEK293 cells stably or transiently expressing hERG constructs were maintained as described previously , 19, 52.Escherichia coli (Invitrogen) were transformed using standard procedures. The mutation was confirmed by sequencing of the entire ORF .The F557L hERG mutation was generated using the Quik Change® site-directed mutagenesis kit . In brief, a pair of complementary oligonucleotide primers containing the mutation  was used in a PCR  by using hERG in a modified pcDNA3.0 vector as a DNA template. A DpnI digest of the PCR mixture was then performed for 1 h at 37 °C. Competent DH5α IhERG recordings, glass shards containing plated cells were placed in a recording chamber mounted on an inverted microscope (Nikon Diaphot). The cells were superfused continuously with Tyrode's solution containing 140 mm NaCl, 4 mm KCl, 2.5 mm CaCl2, 1 mm MgCl2, 10 mm glucose, 5 mm HEPES (titrated to pH 7.4 with NaOH). As described previously (+” version of this solution (containing 94 mm KCl and 50 mm NaCl) was used to obtain recordable currents from comparatively poorly expressed hERG mutants (T623A and F656A). Patch pipettes  were pulled  and polished  to final resistance values between 2 and 4 megaohms. The patch pipettes were filled with intracellular solutio, containing 130 mm KCl, 1 mm MgCl2, 5 mm EGTA, 5 mm MgATP, and 10 mm HEPES (titrated to pH 7.2 with KOH). Pipette resistance compensation was between 70 and 80%. Cavalli-2   . Thus, meviously , 54, 55.IhERG recordings were made using an Axopatch 200B amplifier  and a CV203BU head stage. Data were acquired using a Digidata 1320 interface . Data digitization rates were between 10 and 25 kHz during all voltage protocols, and an appropriate bandwidth between 2 and 10 kHz was set on the amplifier. For WT hERG and most of the mutant channels studied, activating voltage commands to +20 mV were used with tail currents observed at either +40 (for most mutants) or −120 mV (for T623A and F656A)  –20, 33. nditions , 33, 52.supporting information. Statistical comparisons were made using paired or unpaired two-tailed t tests, Wilcoxon matched-pairs signed-rank test, or one-way ANOVA followed by a Bonferroni post-test as appropriate. Details of the statistical test used to evaluate significance for results of particular experiments are given alongside p values under “Results” or in the relevant table or figure legend. p values of < 0.05 were taken as statistically significant.Data analysis was performed using Clampfit 10.3 , Prism versions 4.03 and 5.03, and Excel 2013. Data are presented as the mean ± S.E. or as mean with ±95% CI. Equations used to fit particular data sets are given in the IhERG-drug and IhERG-control are the amplitude of tail currents, respectively, in the presence and absence of a defined concentration of Cavalli-2; the ratio of these two values represented the fraction of unblocked IhERG (see B). Cavalli-2 block reached a steady state within 5 min; therefore, rundown correction was not required.The fractional block (FB) of hERG tail currents by the range of drug concentrations studied was determined using the following equation,hERG see B. Cavall50 and the nH values,x is the concentration expressed as a logarithm, y is the fractional block of IhERG at a given concentration, and IC50 and nH are as defined above.Concentration-response relationships were constructed by plotting mean fractional block of hERG tail currents against concentration of Cavalli-2 and then fitting the experimental data with a standard Hill equation, yielding the ICIhERG activation was established by using the step protocol reported in Fig. 1D. The normalized tail current amplitude was plotted against the command potential of the previous depolarizing step, and the experimental data points were fitted with a Boltzmann function of the following form,I is the tail current elicited after the test voltage x, Imax is the maximal current recorded, V0.5 is the voltage that elicits half-maximal activation, and Slope is the slope factor of the curve. The same equation was used to simulate the I-V curves shown in Fig. 1D.The voltage dependence of DK values for Cavalli-2 inhibition at +60 mV and a reference voltage of 0 mV were estimated and substituted into the following equation,DK+60mV and DK0mV represent half-maximal blocking concentrations at +60 and 0 mV, respectively; V is membrane test potential (+60 mV in this instance); and z, R, F, and T have their usual meanings  (5K7L) (1LNQ) .The following structures and models were used for computational docking and homology model construction. Docking was largely done using the recent cryo-EM open pore structure of hERG (Protein Data Bank code 5VA1) . A homol) (5K7L)  using MO) (5K7L)  with PRO) (5K7L)  to asses) (1LNQ)  because ) (1LNQ) , 20, 29.) (1LNQ)  and hERG) (1LNQ) , we realComputational docking was done using two independent docking methods, GOLD and Flexidock as described previously , 29. FreM. V. H. and C. E. D. data curation; M. V. H., J. C. H., and C. E. D. formal analysis; M. V. H., Y. Z., A. E. H., C. D., J. C. H., and C. E. D. investigation; M. V. H., J. C. H., and C. E. D. writing-original draft; M. V. H., Y. Z., A. E. H., C. D., J. C. H., and C. E. D. writing-review and editing; Y. Z., A. E. H., C. D., J. C. H., and C. E. D. resources; J. C. H. and C. E. D. conceptualization; J. C. H. and C. E. D. supervision; J. C. H. and C. E. D. funding acquisition; J. C. H. and C. E. D. methodology; J. C. H. and C. E. D. project administration.


Late gadolinium enhancement (LGE) is a well-established technique that generates excellent contrast between normal and pathologic myocardium. Selecting the correct inversion time (TI) for nulling normal myocardium is essential for good quality images and accurate diagnosis. The Look-Locker (LL) TI scout is commonly used for TI estimation but is subjective and requires a long breath-hold (20 heartbeats). T1-mapping is a quantitative method that can provide a direct numeric TI prescription in a shorter breath-hold but has not been tested in practice.CMR: 39 subjects  with a variety of cardiac conditions underwent CMR at 1.5T . All received intravenous gadolinium contrast . Imaging included the LL TI scout followed immediately by post-contrast T1-mapping  at a mid-ventricular short-axis slice, and LGE (PSIR) imaging using conventional TI selection based on visual assessment of the TI scout according to radiographer experience.Image processing: matching regions of interest (ROIs) were prescribed on LL TI scout, post-contrast T1-maps and LGE images in an area of the LV without obvious abnormality. LL TI was calculated by linear interpolation of sign reconstructed from the 5-6 lowest SI measurements, while T1-map TI was calculated as TI = 0.69 × T1 from post-contrast T1-maps. We also measured the average SI of the ROI on LGE images to assess for adequate nulling (SI=0) of the myocardium.2=0.77, p < 0.01) between the TI calculated from LL TI scouts and post-contrast T1-maps. The LL TI was 37 ms shorter than T1map TI, consistent with the speed of T1 recovery between measurements , combined with T1 correction for this T1-mapping sequence . In 18% (7/39) of the cases, the operator did not prescribe the correct TI, predominantly when the correct TI is short but the operator overestimated by assuming a starting TI of 300 ms typical for PSIR LGE imaging. Deviation of the prescribed TI from the calculated TI was correlated to suboptimal nulling of the myocardium, whether calculation of the TI was performed using ROIs on LL TI scouts (r2=0.34) or post-contrast T1-maps (r2=0.42). Figure There was excellent correlation (rPost-contrast T1-mapping is an easy way to provide a direct numeric TI for optimal myocardial nulling in a single short breath-hold, reducing operator error and improving robustness for LGE imaging.