Structurally Diverse Bisbenzylisoquinoline Alkaloids with Antiadipogenic Activity through PPARγ Downregulation from the Embryo of Nelumbo nucifera Seeds.

Six undescribed and six known bisbenzylisoquinoline alkaloids were isolated from the embryo of Nelumbo nucifera seeds. Their structures were fully characterized by a combination of 1H, 13C NMR, 2D NMR, and HRESIMS analyses, as well as ECD computational calculations. The antiadipogenic activity of 11 alkaloids was observed in a dose-responsive manner, leading to the suppression of lipid accumulation in 3T3-L1 cells. Luciferase assay and Western blot analysis showed that the active alkaloids downregulated peroxisome proliferator-activated receptor gamma (PPARγ, a key antiadipogenic receptor) expression in 3T3-L1 cells. Analysis of the structure-activity relationship unveiled that a 1R,1'S configuration in bisbenzylisoquinoline alkaloids led to a notable enhancement in antiadipogenic activity. The resistance level against lipid accumulation highlighted a consistent pattern with the suppressive effect on the PPARγ expression. These activity results indicate that alkaloids from the embryo of N. nucifera seeds have a potential of antiobesity effects through PPARγ downregulation.

Silver-dendrimer nanocomposite as emerging therapeutics in anti-bacteria and beyond.

To develop next-generation nanomedicine, theranostic nanotherapeutic strategies are increasingly being emphasized. In recent years, it is observed that the effective lifetime of anti-bacterial and anti-cancer agent is diminishing, which undermines the economic incentives necessary for clinical development and therapeutic applications. Thus, novel formulations ought to not only kill drug resistant strains and cancerous cells but also inhibit their formation. Recently, metallic nanoparticles [for example- silver (Ag) nanoparticles] have been widely investigated for their biomedical applications. The so-called applications necessitate the inclusion of these nanoparticles inside polymeric matrices (for example- dendrimer) leading to chemical functionalization of the metallic nanoparticles. Silver and silver nanoparticles' antibacterial activity has already been well established over years. Dendrimers due to their homogeneous highly branched structure and uniform composition are perfectly suitable for the inclusion of silver nanoparticles [Ag NPs]. Recently, the increasing trend in the development of Ag-dendrimer nanocomposites is attributed to the excellent antibacterial activity of Ag as well as dendrimer's unique properties like variable functional terminal ends and potential antibacterial effect necessarily. This review provides an informative overview regarding the numerous aspects of bactericidal and other biomedical applications of Ag-dendrimer nanocomposites, particularly emphasizing analysis of existing research and prospective worth to the pharmaceutical sector in future.

3D-printing magnesium-polycaprolactone loaded with melatonin inhibits the development of osteosarcoma by regulating cell-in-cell structures.

Melatonin has been proposed as a potent anticarcinogen presents a short half-life for osteosarcoma (OS). Cell-in-cell (CIC) structures play a role in the development of malignant tumors by changing the tumor cell energy metabolism. This study developed a melatonin-loaded 3D printed magnesium-polycaprolactone (Mg-PCL) scaffold and investigated its effect and molecular mechanism on CIC in OS. Mg-PCL scaffold was prepared by 3D-printing and its characteristic was determined. The effect and molecular mechanism of Mg-PCL scaffold as well as melatonin-loaded Mg-PCL on OS growth and progression were investigated in vivo and in vitro. We found that melatonin receptor 1 (MT1) and CIC expressions were increased in OS tissues and cells. Melatonin treatment inhibit the key CIC pathway, Rho/ROCK, through the cAMP/PKA signaling pathway, interfering with the mitochondrial physiology of OS cells, and thus playing an anti-invasion and anti-metastasis role in OS. The Mg-PCL-MT could significantly inhibit distant organ metastasis of OS in the in vivo model. Our results showed that melatonin-loaded Mg-PCL scaffolds inhibited the proliferation, invasion and metastasis of OS cells through the CIC pathway. The Mg-PCL-MT could be a potential therapeutics for OS.

TLC-MS identification of alkaloids in Leonuri Herba and Leonuri Fructus aided by a newly developed universal derivatisation reagent optimised by the response surface method.

INTRODUCTION: Dragendorff's reagent has low sensitivity and non-specificity for some alkaloids. A new alkaloid derivatisation reagent has been developed and optimised by using a Box-Behnken design method. This new reagent is applicable for structurally diverse natural alkaloids, and is proposed as a universal alkaloid staining reagent. OBJECTIVE: To establish an efficient and sensitive thin-layer chromatography (TLC) identification method for Leonuri Herba and Leonuri Fructus to characterise their differences and similarities. METHOD: Three key components (bismuth subnitrate, potassium iodide and iodine) in the derivatisation reaction were re-constructed and optimised using a response surface method. Different inorganic acids, essential additives of the staining reaction, were compared by a single-factor experiment design. RESULTS: This newly optimised reagent for alkaloids reported in this study, named the enhanced Dragendorff-Wagner's reagent, is composed of 0.82% bismuth subnitrate, 11.1% potassium iodide and 0.76% iodine in 38% phosphoric acid solution. Validation results indicate that the TLC spot of stachydrine stained with the enhanced Dragendorff-Wagner's reagent had a limit of detection of 2.0 µg, good intra- and inter-plate, and intra- and intra-day precisions with relative standard devition values less than 5.0%, and stability over 90 min. CONCLUSION: This enhanced Dragendorff-Wagner's reagent was applied for TLC identification of 16 reference alkaloids representing 11 structural skeletons, and two closely related herbs (Leonuri Herba and Leonuri Fructus). This newly enhanced Dragendorff-Wagner's reagent is a universal, effective, and sensitive staining reagent for alkaloids.

Quantitative 1 H nuclear magnetic resonance (qHNMR) methods for accurate purity determination of glucosinolates isolated from Isatis indigotica roots.

INTRODUCTION: Glucosinolates (1-5) are important secondary metabolites found in Isatis indigotica roots. Due to their high hydrophilic and ionic nature, purified glucosinolates often contain salt impurities and moisture. Accurate assessment of their purities is important for glucosinolates being utilised as chemical markers. OBJECTIVE: To develop and validate quantitative proton (1 H) nuclear magnetic resonance (qHNMR) methods for purity assessments of aliphatic and indole glucosinolates (1-5). METHOD: Several NMR parameters such as pulse program, relaxation time, and delay time were optimised. Three qHNMR methods were developed using gluconapin (3), neoglucobrassicin (4), and sinigrin (5) for method validation and with maleic acid as internal standard. RESULTS: The quantification was based on the integrated area ratios of an olefinic proton (H-4 for 1-3; H-6 for 4; and H-3 for 5) of the side chain from glucosinolates relative to the olefinic proton from the internal standard using deuterated water (D2 O) as the solvent. The qHNMR methods were successfully applied for purity assessments of four aliphatic glucosinolates (1-3 and 5: progoitrin, epiprogoitrin, gluconapin, and sinigrin), and an indole glucosinolate (4: neoglucobrassicin). CONCLUSION: The purity of glucosinolates isolated from I. indigotica and commercial sinigrin was accurately assessed using the developed qHNMR method. The qHNMR provides a reliable and superior means to determine the purity of glucosinolates.

Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.

Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination.

Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.

Improved TLC Bioautographic Assay for Qualitative and Quantitative Estimation of Tyrosinase Inhibitors in Natural Products.

INTRODUCTION: TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays. OBJECTIVE: An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study. METHODS: L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent. RESULTS: The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-ß-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay. CONCLUSION: Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

[Anti-complement alkaloids from whole plants of Viola yedoensis].

Fifteen alkaloids were isolated from the 95% ethanol extract of the whole plants of Viola yedoensis by various column chromatographic techniques such as silica gel and Sephadex LH-20. Their structures were identified as neoechinulin A（1）,N-benzoyl-L-p-hydroxy-phenylalaninol（2）,aurantiamide acetate（3）,aurantiamide（4）,anabellamide（5）,trichosanatine（6）,indole-3-carboxylic acid methyl ester（7）,3-carboxyindole（8）,N-trans-feruloyl-tyramine（9）,paprazine（10）,7'-（3', 4'-dihydroxyphenyl）-N-[(4-methoxyphenyl)ethyl]propenamide（11）,cannabisin F（12）,N-(4-hydroxyphenethyl)octacosanamide（13）,N-(4-hydroxyphenethyl)hexacosanamide（14）and N-benzoyl-L-phenylalaninol（15）. All the compounds except 3 and 4 were isolated from this plant for the first time. These alkaloids exhibited anti-complement activity against the classical pathway(CP)and the alternative pathway(AP)with the CH50 and AP50 values ranging from 0.12 to 0.33 g•L⁻¹ and 0.22 to 0.50 g•L⁻¹, respectively. Preliminary mechanism study using complement-depleted sera showed that these alkaloids acted on different complement components in the complement activation cascade.

Needle Trap Device as a New Sampling and Preconcentration Approach for Volatile Organic Compounds of Herbal Medicines and its Application to the Analysis of Volatile Components in Viola tianschanica.

INTRODUCTION: The needle trap device (NTD) technique is a new microextraction method for sampling and preconcentration of volatile organic compounds (VOCs). Previous NTD studies predominantly focused on analysis of environmental volatile compounds in the gaseous and liquid phases. Little work has been done on its potential application in biological samples and no work has been reported on analysis of bioactive compounds in essential oils from herbal medicines. OBJECTIVE: The main purpose of the present study is to develop a NTD sampling method for profiling VOCs in biological samples using herbal medicines as a case study. METHODOLOGY: A combined method of NTD sample preparation and gas chromatography-mass spectrometry was developed for qualitative analysis of VOCs in Viola tianschanica. A 22-gauge stainless steel, triple-bed needle packed with Tenax, Carbopack X and Carboxen 1000 sorbents was used for analysis of VOCs in the herb. Furthermore, different parameters affecting the extraction efficiency and capacity were studied. RESULTS: The peak capacity obtained by NTDs was 104, more efficient than those of the static headspace (46) and hydrodistillation (93). This NTD method shows potential to trap a wide range of VOCs including the lower and higher volatile components, while the static headspace and hydrodistillation only detects lower volatile components, and semi-volatile and higher volatile components, respectively. CONCLUSION: The developed NTD sample preparation method is a more rapid, simpler, convenient, and sensitive extraction/desorption technique for analysis of VOCs in herbal medicines than the conventional methods such as static headspace and hydrodistillation. Copyright © 2016 John Wiley & Sons, Ltd.

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

INTRODUCTION: Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. OBJECTIVE: To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. METHODS: The new TLC bioautographic assay was based on reaction of lipase with ß-naphthyl myristate and the subsequent formation of the purple dye between ß-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. RESULTS: The ß-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). CONCLUSION: The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.

Anti-Complementary Components of Helicteres angustifolia.

A first phenalenon derivative with an acetyl side chain at C-8, 8-acetyl-9-hydroxy-3-methoxy-7-methyl-1-phenalenon (compound 1), and a pair of new sesquilignan epimers at C-7″ of hedyotol C and hedyotol D analogs, hedyotol C 7″-O-ß-d-glucopyranoside (compound 2) and hedyotol D 7″-O-ß-d-glucopyranoside (compound 3) were isolated from the aerial parts of Helicteres angustifolia together with nine known compounds (4-12). Their structures were elucidated on the basis of spectroscopic methods, including mass spectroscopy, and 1D and 2D nuclear magnetic resonance. Eleven isolates exhibited anti-complementary activity. In particular, compounds 4 and 5 exhibited potent anti-complementary activities against the classical and alternative pathways with CH50 values of 0.040 ± 0.009 and 0.009 ± 0.002 mM, and AP50 values of 0.105 ± 0.015 and 0.021 ± 0.003 mM, respectively. The targets of compounds 4 and 5 in the complement activation cascade were also identified. In conclusion, the anti-complementary components of H. angustifolia possessed chemical diversity and consisted mostly of flavonoids and lignans in this study.

Flavonol glycosides and other phenolic compounds from Viola tianshanica and their anti-complement activities.

CONTEXT: Viola tianshanica Maxim. (Violaceae) is a perennial herb distributed in Central Asia, especially in the Xinjiang Uygur Autonomous Region (XUAR) of China. Preliminary study showed that the ethanol extract of the herb exhibited the anti-complement activity against the classical pathway, but the active components responsible for this capacity remain unknown and are yet to be studied. OBJECTIVE: The objective of this study was the isolation and identification of the anti-complement constituents of V. tianshanica. MATERIALS AND METHODS: The ethyl acetate and n-butanol fractions from the ethanol extract of V. tianshanica were purified. The structures of the isolates were identified by spectroscopic methods, and comparing their spectral data with those reported in the literature. All the isolates (0.02-2.50 mg/mL) were evaluated for their anti-complement activity against the classical and alternative pathways. RESULTS: Twenty-one phenolic compounds including 15 flavonol O-glycosides (1-15), one flavone 6,8-di-C-glycoside (16), one flavone aglycone (17), and four phenolic acid derivatives (18-21) were isolated and identified. Bioassay showed that 11 compounds inhibited the classical pathway and the alternative pathway with CH50 and AP50 values of 0.113-1.210 mM and 0.120-1.579 mM, respectively. Preliminary mechanistic study using complement-depleted sera demonstrated that 1 acted on C1q, C2, C4, and C9 components, 16 on C1q, C4, and C5, and 21 on C1q, C3, C4, and C9. CONCLUSION: All isolated compounds except 1 and 10 were reported for the first time from V. tianshanica. Compound 16 is the first flavone C-glycoside isolated from the herb. Flavonol O-glycosides and phenolic acids contributed the anti-complement activity of the herb.

Crystal structure of a Tankyrase-Axin complex and its implications for Axin turnover and Tankyrase substrate recruitment.

Axin is a tumor suppressor and a key negative regulator of the Wnt/ß-catenin signaling pathway. Axin turnover is controlled by its poly-ADP-ribosylation catalyzed by tankyrase (TNKS), which requires the direct interaction of Axin with TNKS. This interaction is thus an attractive drug target for treating cancers, brain injuries, and other diseases where ß-catenin is involved. Here we report the crystal structure of a mouse TNKS1 fragment containing ankyrin-repeat clusters 2 and 3 (ARC2-3) in a complex with the TNKS-binding domain of mouse Axin1. Surprisingly, we found that Axin contains two discrete TNKS-binding segments, both of which bind simultaneously to the two ARC2 domains in the ARC2-3 homodimer. Our crystal structure shows that in each TNKS-binding segment of Axin there is a conserved glycine residue that lies in the bottom of a narrow "gate" formed by two parallel tyrosine side chains on the TNKS surface. This glycine-selection gate is crucial for TNKS-Axin interactions, as mutation of the TNKS gate-forming residues, or mutation of either glycine residue in the two Axin segments, completely abolishes the binding of the corresponding Axin segment to TNKS. The bivalent binding of Axin to TNKS is required for Axin turnover, since mutations in either gate-binding glycine residue in Axin lead to Axin stabilization in the cell. In addition, our analyses also reveal the structural basis for TNKS substrate recruitment, and shed light on the overall structure of TNKS that should help in developing specific inhibitors of Wnt/ß-catenin signaling.

Anticomplement monoterpenoid glucosides from the root bark of Paeonia suffruticosa.

Six new (1-6) and 19 known monoterpenoid glucosides were isolated from the root bark of Paeonia suffruticosa. The monoterpenoid glucosides 1, 2, 7, 10-19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. In a mechanistic study, suffrupaeoniflorin A (1) interacted with C1q, C3, C5, and C9, while galloylpaeoniflorin (12) and galloyloxypaeoniflorin (19) acted on C1q, C3, and C5 components in the complement activation cascade.

Anti-bacterial cyclic dipeptides from Hormonema dematioides, an endophytic fungus of Juniperus communis.

Hormonema sp., an endophytic fungi found in the medicinal plant of Juniperus communis leaves, was reported to possess antimicrobial compounds from its unidentified species. In this study, 21 cyclic dipeptides (1-21) were isolated and identified from H. dematioides. All the 21 isolated cyclic dipeptides were reported for the first time from the genus Hormonema. The antimicrobial activity by the disc diffusion method showed that five compounds including cyclo(Pro-Gly) (9), cyclo(Phe-Ile) (11), cyclo(Ile-Val) (12), cyclo(Val-Ala) (17), and cyclo(Ala-Phe) (20) inhibited the growth of Staphylococcus aureus with inhibition zone diameters ranging from 12 to 30 mm. Further bioassay demonstrated that four cyclic dipeptides (9, 12, 17, and 20) showed significant antibacterial activity against S. aureus with MIC values of 0.04, 0.39, 0.01, and 0.10 µg/mL, respectively, as compared to the positive control (ciprofloxacin, MIC = 0.08 µg/mL). However, none of these cyclic dipeptides showed obvious anti-fungal activity against Candida albicans.

Enhancing lathyrane structural diversity and MDR activity by combinatorial modification of lathyrane nucleus and ester side chain: A case study of Euphorbia Factor L1 and Euphorbia Factor L3.

The chemical transformation of lathyrane nucleus through reduction and oxidation reactions using Euphorbia Factor L1 (EFL1) and Euphorbia Factor L1 (EFL3) as examples were investigated, along with a co-modification strategy of lathyrane nucleus and its side ester chain. A total of 38 lathyrane derivatives (5-42) including 34 new compounds were obtained, which greatly enriched the structural diversity of the lathyrane-type diterpenoids. Cytotoxicity against drug-sensitive and drug (adriamycin, ADM) resistant MCF-7 cells showed that 23 out of 38 transformed derivatives possessed obvious cytotoxic activity with IC50 values ranging from 7.0 to 41.1 µM and 3.2 to 45.5 µM, respectively, against both cells, compared to the noncytotoxic EFL1 and EFL3. The multidrug resistance (MDR) reversing activities of these lathyrane derivatives were further evaluated in MCF-7/ADM. Three transformed compounds (reversal fold, RF = 151.33, 62.94 and 47.3 for 27, 37 and 42) showed markedly higher activity than EFL1 (RF = 32.92) and EFL3 (RF = 39.68). Structure-activity relationship study revealed an essential role of C-6/17 and C-12/13 double bonds on lathyrane nucleus for exerting MDR reversal activity. Western blotting analysis showed that 42 could reduce the expression level of P-glycoprotein (P-gp) in MCF-7/ADM cells; however, the most active compound 27 with an unnatural 5/7/7/4 fused-ring diterpenoid skeleton, had no inhibitory effect on P-gp expression.

Rat Model of Neuropathic Pain Induced by Spinal Nerve Ligation: A New Approach via an Oblique Lateral Incision.

Purpose: The spinal nerve ligation (SNL) model is a typical peripheral neuropathic pain model. During its construction, the removal of paraspinal muscles and transverse processes typically occurs, resulting in additional trauma that may potentially affect the pathophysiologic process of neuropathic pain. This study aimed to investigate the feasibility of establishing a more reliable SNL model using an oblique lateral approach. Methods: 36 adult male Sprague-Dawley rats were randomly divided into three groups: the traditional SNL (T-SNL) group, the new SNL (N-SNL) group (where the left L5 spinal nerve was ligated with a titanium clip via an oblique lateral approach), and the sham-operated (Sham) group. The operation time, Intraoperative bleeding, the number of rats that died, gait behavior, mechanical and cold pain threshold were recorded and measured. Stereology technology was used to calculate the number of microglia in spinal dorsal horn, and the Enzyme-linked immunosorbent assay (ELISA) technology was used to detect the expression of TNF-α and IL-1ß in spinal cord as well as C-reactive protein (CRP) in serum in order to assess the effect of surgery on animal inflammation. Results: Compared with the T-SNL group, operative time and intraoperative bleeding were significantly decreased in the N-SNL group. Within 14 days postoperation, one rat in the N-SNL group was died, two rats in the T-SNL group were died. Compared with the Sham group, the N-SNL group showed obvious spontaneous pain behavior, decreased the pain thresholds, the number of microglia and the expression of TNF-α and IL-1ß were significantly increased, and there was no significant difference in these indexes compared with T-SNL group. There was no significant difference in serum CRP levels among the three groups. Conclusion: This study suggests that the oblique lateral approach SNL model is a reliable NP model with the advantages of good reproducibility, accessibility, and low trauma.

Structural basis for translational inhibition by the tumour suppressor Pdcd4.

Pdcd4 is a tumour suppressor protein. It inhibits translation through interaction with translation initiator eIF4A, resulting in the suppression of neoplastic transformation and tumour invasion. Here, we present the crystal structures of an N-terminal-truncated Pdcd4 in free form and in complex with eIF4A. Upon binding to eIF4A, Pdcd4 undergoes a marked conformational change to form a heterotrimeric complex with eIF4A, with one Pdcd4 binding to two eIF4A molecules in two different modes. The binding of Pdcd4 to eIF4A is required to inhibit the enzymatic activity of eIF4A, translation initiation, and AP-1-dependent transcription. Both MA3 domains are required to efficiently compete with the C-terminal domain of eIF4G (eIF4Gc) for binding to eIF4A whereas a single MA3 is sufficient to inhibit translation. Our structural and mutational analyses reveal that Pdcd4 inhibits translation initiation by trapping eIF4A in an inactive conformation, and blocking its incorporation into the eIF4F complex.