These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits TNF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein with redox activity and is proved to be secreted from stimulated cells. The aim of this study was to evaluate the functions of extracellular APE1/Ref-1 with respect to leading anti-inflammatory signaling in TNF-α-stimulated endothelial cells in response to acetylation. Treatment of TNF-α-stimulated endothelial cells with an inhibitor of deacetylase that causes intracellular acetylation, considerably suppressed vascular cell adhesion molecule-1 (VCAM-1). During TSA-mediated acetylation in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human (rh) APE1/Ref-1 with reducing activity induced a conformational change in rh TNF-α receptor 1 (TNFR1) by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1, in accordance with the activation of p66 Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis and other vascular diseaseIn vascular inflammatory responses, TNF-α which is released from macrophages exert direct effects on a multitude of secondary inflammatory mediators via binding with the TNF-α receptors (mainly TNFR1)611Apurinic apyrimidinic endonuclease 1/Redox factor-1  is a multifunctional protein; its N-terminal region is involved in redox activity and regulates multiple transcription factors, and its C-terminus is involved in base excision DNA repair activity1516Because the level of secreted APE1/Ref-1 is substantially increased in response to acetylation, we hypothesized that secreted APE1/Ref-1 could be an effective regulator in inflammatory reactions via its reduction. We tested this hypothesis using TNF-α-treated human umbilical vein endothelial cells (HUVECs) as a vascular inflammation model. We provide compelling experimental evidence to indicate that extracellular secreted APE1/Ref-1 in response to intracellular acetylation inhibits inflammatory signaling via a reduction in TNFR1, showing that treatment of anti-APE1/Ref-1 antibody in histone deacetylase inhibitor (HDACi), trichostatin A (TSA)-mediated modulation against TNF-α-stimulated endothelial activation recovers not only upregulation of adhesion molecule but also the generation of ROS.The HDACi, TSA inhibits the expression of the cell adhesion molecule VCAM-1 in TNF-α-stimulated endothelial cellsTo test whether TSA-mediated acetylation causes downregulation of VCAM-1 in TNF-α-stimulated endothelial cells, we observed the effect of deacetylase on VCAM-1 levels using an adenovirus expressing HDAC3. The level of VCAM-1 in TNF-α-treated cells was substantially increased in HDAC3-overexpressed cells presenting no acetylation . As showIn previous studies, we reported that acetylated APE1/Ref-1 (Ac-APE1/Ref-1) is secreted after intracellular acetylation in TSA-treated human embryonic kidney epithelial cells (HEK 293 T)To confirm whether APE1/Ref-1 secretion is regulated by acetylation, we examined the acetylation of secreted APE1/Ref-1 using anti acetyl-lysine antibody. TSA-mediated acetylation caused an initial increase in secreted Ac-APE1/Ref-1 compared with untreated control cells . InteresTo further examine the functional role of extracellular secreted APE1/Ref-1, we determined the effect of the anti-APE1/Ref-1 antibody on VCAM-1 expression in TNF-α-stimulated HUVECs. VCAM-1 was not detected after treatment with only TSA, IgG or anti-APE1/Ref-1, but was markedly increased in TNF-α-stimulated cells . As showIntracellular ROS generation is involved in inflammatory signaling in TNF-α-stimulated HUVECsAdditionally, a change in mitochondrial ROS generation in response to TSA-mediated acetylation was observed using the mitochondrial superoxide indicator, MitoSox in TNF-α-stimulated endothelial cells. As shown in Next, we raised the question of whether secreted APE1/Ref-1 showed a redox function after deacetylation, leading to the regulation of TNFR1-mediated inflammatory signaling. We determined the redox activity of recombinant human APE1/Ref-1 (rh APE1/Ref-1) by measuring luminescent intensity, which was generated by luciferin formation after substrate reduction and reacted with luciferase, in a mixture containing the reductase substrate proluciferin and reductase rh APE1/Ref-1. The redox activity was proportional to the concentration of rh APE1/Ref-1 . MoreoveBased on the observation that rh APE1/Ref-1, but not rh Ac-APE1/Ref-1, increased luminescent intensity, indicating reductive activity, we hypothesized that secreted APE1/Ref-1 can reduce other oxidized proteins, para- and autocrinally, in the culture supernatant after secretion. To evaluate the role of extracellular secreted APE1/Ref-1, we observed whether rh APE1/Ref-1 directly reduced rh TNFR1 containing 12 disulfide bonds via oxidization of free thiol groups. We performed a modified biotin-switch assay using recombinant TNFR1 as a substrate. As shown in Shc and MAPKs was investigated. As shown in shc on serine 36 residue increased in TNF-α-treated endothelial cells and then considerably decreased by 50% after TSA treatment. Consistent with the results for ROS generation, suppressed p66Shc was almost completely reactivated after treatment with a neutralizing APE1/Ref-1 antibody, to a level similar to that of TNF-α treated cells, although immunoglobulin did not affect the reactivation of p66shc. Additionally, the contribution of MAPK activation to TNF-α-induced Ser-phosphorylation of p66Shc was studied. When TNF-α- and TSA-treated cells were incubated with neutralizing APE1/Ref-1 antibody, the suppressed phosphorylation of p66shc increased significantly (p < 0.001 vs. TNF-α-/TSA-treated cells) as shown in shc in TNF-α/TNFR1 inflammatory signaling did not observed (data not shown). Thus, these results suggest that p66Shc and p38, but not JNK or ERK, are involved in anti-inflammatory signals via secreted APE1/Ref-1 in TNF-α-stimulated cells.To gain insight into the mechanism of secreted APE1/Ref-1-mediated suppression of TNF-α/TNFR1 inflammatory signaling, the involvement of p66Shc and p38 MAPK in TSA-mediated acetylated cells were recovered by treatment with a neutralizing anti-APE1/Ref-1 antibody; (d) rh APE1/Ref-1 caused a conformational change in the extracellular domain of TNFR1, showing substantial reducing ability in biochemical assays. Because the acetylation-induced anti-inflammatory effects in TNF-α-stimulated endothelial cells was inhibited by pretreatment with the neutralizing antibody, it is reasonable to conclude that secreted APE1/Ref-1, but not the acetylated form, initially regulates inflammatory signals via disruption of TNFR1, preventing binding with TNF-α.An understanding of the function of extracellular APE1/Ref-1 is necessary for its application as an anti-inflammatory agent and to establish clinical trials to support a mechanism-based approach to treat vascular inflammatory disease. The present study provides novel evidence for the role of the auto- or paracrine secretory protein in inflammation and indicates that the anti-inflammatory regulation of secreted APE1/Ref-1 in TNF-α-stimulated endothelial cells after TSA treatment is tightly linked to the inactivation of TNF-α/TNFR1 signaling. This conclusion is based on the following observations: (a) TSA-mediated acetylation causes the secretion of Ac-APE1/Ref-1 in TNF-α-stimulated endothelial cells, which show a substantial decrease in VCAM-1 expression; (b) secreted Ac-APE1/Ref-1 was rapidly deacetylated; (c) not only the suppression of VCAM-1 expression and ROS generation, but also the inactivation of p66in vitro and in vivo, even though the molecular basis for this effect is unclear. Moreover, acetylation at a specific amino acid residue, i.e., lysine, and changes in the function of cellular proteins have also been reported, including changes in DNA-protein interactions, transcriptional activity, protein stability, and enzymatic activityin vitro tumor cell invasivenessAcetylation, a type of post-translational modification, is considered an important mechanism in the regulation of gene expression via activation of nuclear histone proteins; it regulates the functions of multiple cytoplasmic proteins involved in cell survival, proliferation, and death, as well as disease-related intracellular signaling. Cellular acetylation increases rapidly via HDACi administration. HDAC inhibitors are potential chemotherapeutic agents for cancer and inflammatory diseases2627Our observation that APE1/Ref-1 was secreted in TNF-α-stimulated endothelial cells after TSA-mediated hyperacetylation strongly supports the functional role of extracellular APE1/Ref-1. The secreted and acetylated APE1/Ref-1 showed no redox activity owing to conformational changes via acetylation according to the results of biochemical assays. However, the Ac-APE1/Ref-1 could be rapidly converted to its native form by removing the acetyl moiety from the ε-amino groups of the protein substrate lysine residue, indicating the recovery of reducing activity. APE1/Ref-1 contains two cysteine residues (cysteines 65 and 93) in its redox active siteN-acetylcysteine can cleave disulfide bonds, resulting in a change in protein structure, and hence a change in functionshc/MAPK, and VCAM-1 expression are induced by secreted APE1/Ref-1, either auto- or paracrinally. However, further studies are needed to systematically explore this possibility, including the identification of molecules that promote the interaction with the substrate TNFR1.In TNF-α-stimulated inflammation, TNFR1 is a potentially selective substrate for secreted APE1/Ref-1. TNFR1 is characterized by the ability to bind TNF-α via extracellular CRDs; the receptor itself is comprised of a chain of four CRDs, each of which is stabilized by three pairs of disulfide-linked cysteine residuesIt is plausible that multiple disulfide bonds in the extracellular domain of other transmembrane receptors can be affected by reducing proteins. Cytokines and biochemical mediators released during inflammation intensify and propagate the inflammatory response. These mediators can act systemically and activate other receptors simultaneously. Based on our studies, inflammatory reactions stimulated by cytokines through the IL-1 receptor or the Toll-like receptor (each has 5 disulfide bonds in the extracellular domain) were effectively attenuated by exposure to APE1/Ref-1  and 5. IIn conclusion, the present study offers novel insight into the mechanisms of hyperacetylation-mediated anti-inflammation signals in human TNF-α-endothelial cells and shows a strong relationship between secreted APE1/Ref-1, but not Ac-APE1/Ref-1, and TNF-α/TNFR1 and the inflammatory process. Based on these observations, we propose that rh APE1/Ref-1 with reducing activity may be a useful therapeutic agent for the treatment of cytokine responses involved in vascular inflammation. Clarifying the mechanisms of the post-translational modification of secreted APE1/Ref-1 may provide specific markers for vascular diseases and facilitate the development of a vascular inflammation inhibitor.2.HUVECs were cultured in endothelial growth medium as previously reportedAdenoviruses encoding β-galactosidase , full-length APE1/Ref-1 (AdAPE1/Ref-1), and HDAC3 (AdHDAC3) were preparedHUVECs were pretreated with TSA for the indicated time periods and concentrations followed by treatment with 15 ng/ml TNF-α for 12 h. The secreted acetylated form, APE1/Ref-1 in the culture supernatant was chemically pulled-downThe effects of secreted APE1/Ref-1 on intracellular ROS generation or mitochondrial superoxide production in TNF-α-stimulated endothelial cells were analyzed as described previously with modificationsA sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify the level of secreted APE1/Ref-1 in the culture supernatant as previously describedPurified rh APE1/Ref-1 was prepared as previously reportedWhole cell lysates were prepared and subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotting as described in the All statistical tests were implemented in GraphPad Prism v.5.01  software. Statistical significance in the differences in measured variables between the control and treated groups was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s multiple comparison tests. Differences were considered significant at P < 0.05.How to cite this article: Park, M. S. et al. Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor. Sci. Rep.6, 23015; doi: 10.1038/srep23015 (2016).


Although acupuncture therapy is widely used in traditional Asian medicine for the treatment of diverse internal organ disorders, its underlying biological mechanisms are largely unknown. Here, we investigated the functional involvement of acupuncture stimulation (AS) in the regulation of inflammatory responses. TNF-α production in mouse serum, which was induced by lipopolysaccharide (LPS) administration, was decreased by manual acupuncture (MAC) at the zusanli acupoint . In the spleen, TNF-α mRNA and protein levels were also downregulated by MAC and were recovered by using a splenic neurectomy and a vagotomy. c-Fos, which was induced in the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV) by LPS and electroacupuncture (EAC), was further increased by focal administration of the AMPA receptor blocker CNQX and the purinergic receptor antagonist PPADS. TNF-α levels in the spleen were decreased by CNQX and PPADS treatments, implying the involvement of inhibitory neuronal activity in the DVC. In unanesthetized animals, both MAC and EAC generated c-Fos induction in the DVC neurons. However, MAC, but not EAC, was effective in decreasing splenic TNF-α production. These results suggest that the therapeutic effects of acupuncture may be mediated through vagal modulation of inflammatory responses in internal organs. Acupuncture therapy has been used to treat diverse disorders of the internal organ in several Asian countries, and its use is increasing in Western countries as well . AccordiAmong several biological correlates that may explain the principle of acupuncture, the autonomic nervous system (ANS) is frequently considered to be a mediator of AS because it can interconnect external somatosensory inputs with internal organ responses via the central neural networks ,3. Of thVagus nerve stimulation (VNS) has been demonstrated to reduce inflammatory responses in animals given lipopolysaccharide (LPS) ,13. In aBalb/c mice  were maintained in an animal room with regulated temperature (22°C), 60% humidity, and a 12-hour light/12-hour dark cycle. Animals were allowed to eat commercial chow and to drink water ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols involving preopertative, operative, and postoperative animal care were approved by the Daejeon University Institutional Animal Use and Care Committee and were in accordance with the Animal-use Statement and Ethics Committee Approval Statement for Animal Experiments provided by Daejeon University . None of animals became ill and died during the study. Postoperative animals were euthanized by overdose of ketamine and zylazine (80 mg/kg of ketamine and 5 mg/kg of xylazine). All surgical procedures were conducted in a room under well-ventilated, sterile conditions, and all efforts were made to minimize suffering.Escherichia coli 0127:B8  intraperitoneally (60 μg/kg or 15 mg/kg). Blood was sampled from the tail at 30, 60, 90, 120 and 180 minutes after LPS administration.Endotoxemia was induced in the mice by administering LPS from For manual acupuncture (MAC) under anesthesia, mice were administered 80 mg/kg of ketamine and 5 mg/kg of xylazine, and the needle  was inserted bilaterally into the zusanli acupoint . Acupuncture needles were inserted for 30 minutes with a slow rotation every 5 minutes. For electroacupuncture (EAC) at ST36, electric stimulation was given at 1 volt, 1 Hz, with a 2-msec pulse duration for 30 min by using an isolated pulse stimulator . For the acupuncture experiment using unanesthetized animals, mice were placed and immobilized in 50-ml conical tubes and AS was given in the same way as it was for the anesthetized animals.2SO4. Values of the optical density of the samples were read at 450 nm by using a spectrophotometer .Blood was centrifuged at 800 g for 10 minutes, and the supernatants were collected for TNF-α analysis in plasma. ELISA was performed as recommended by the manufacturer . Briefly, to a 96 well plate pretreated with coating buffer was added 100 μl of capture antibody (anti-mouse TNF-α) in coating buffer. The plate was sealed and incubated overnight at 4°C. Wells were aspirated and washed 5 times with 200 μl of a washing buffer ) and were blocked with 200 μl of assay diluent solution . After incubation at room temperature for 1 hour, 100 μl of standard recombinant mouse TNF-α along with the samples were added to the appropriate wells, and the wells were incubated at room temperature for 2 h. Then, 100 μl of detection antibody (anti-mouse TNF-α) in assay diluents were added to each well, and the wells were incubated at room temperature for 1 h. Finally, 100 μl of avidin-horseradish peroxidase (HRP) diluted in assay diluents were added, and the wells were incubated at room temperature for 30 min to activate the substrate solution. The reaction was stopped by using 2N H™ dye, dNTP mix, optimized buffer), and 1X mouse GAPDH primer , endogenous control (VIC®/MGB probe primer), and PCR = grade water were mixed in a 20 μl of reaction volume. Plate was covered with a film  and was centrifuged at 2,900 rpm  for 5 min to spin down samples. PCR was carried out using an instrument  by activating Taq polymerase for 2 min at 50°C and for 10 min at 95°C, followed by 40 cycles with 15 s at 95°C (denaturation) and 1 min at 60°C . The results were shown as a ratio of TNF-α mRNA to GADPH reference mRNA. The primer sequences used for real-time PCR for TNF-α mRNA were the forward primer (5’-CATCTTCTCAAAATTCGAGTGACAA-3’) and the reverse primer (5’- TGGGAGTAGACAAGGTACAACCC-3’). Values of relative quantitation (RQ) of TNF-α mRNA expression relative to GAPDH reference mRNA was calculated by a software converting program (Applied Biosystems) for threshold cycle (Ct) data.Total RNA was isolated from the spleen by using Easy-BLUE reagent . A reaction for cDNA synthesis was carried out in 30 μl using total RNA (1 μg) as a template, 1X reaction buffer ), 104 μM dNTP), RNasin (30 U), random primer (16 μM Promega), MMLV reverse transcriptase , for 2 h at 37°C. Real-time PCR was performed as follows. Synthesized cDNA (300 ng), 1X Power SYBR Green PCR Master mix , 0.15 μM of forward and reverse TNF-alpha primers, and PCR-grade water in a 20 μl of reaction volume were added to each well of 96-well plate . For the synthesis of a mouse GAPDH reference gene, cDNA (300 ng), 1x TaqMan Gene Expression Master Mix  and the reverse primer (5’-CAGGTACATGGGCTCATACC-3’) for TNF-α mRNA, and the forward primer (5’-CACACTGTGCCCATCTATGA-3’) and the reverse primer (5’-TACGGATGTCAACGTCACAC-3’) for actin mRNA. Amplified DNA sizes were 497 bp and 409 bp for TNF-α and actin, respectively. PCR-amplified DNA was analyzed on agarose gels, and the band intensities from the captured images were quantified using i-Solution software .For RT-PCR analysis, 30 cycles of amplification were optimal for a quantitative comparison of the TNF-α mRNA expression among the samples. The primer sequences used for PCR were the forward primer (Spleen and brain tissues were embedded and frozen at –20°C. Sections (15 or 20 μm thickness) were cut on a cryostat, mounted on positively charged slides, and subjected to immunostaining as described previously [For quantification of the TNF-α signal from spleen tissues, the fluorescence images in the slide were captured by fluorescence microscope coupled with digital camera and transferred to the i-Solution software program (Image and Microscope Technology). Digital images for analysis were taken under the same exposure conditions among experimental groups and no modifications in the brightness and contrast were made for the numerical measures of pixel intensity. Three to five microscopic fields (field size: 410 x 330 mm) were randomly taken from each section and the average value from 4 nonconsecutive sections per each animal was used for statistical comparison among experimental groups. For the quantification of c-Fos signals in the NTS and DMV areas in the horizontal brain sections, digital images captured from the fluorescence microscope under the same exposure conditions were transferred to Adobe Photoshop software. Boundaries of NTS and DMV areas were identified by Hoechst nuclear counterstaining and marked on the images, and individual c-Fos signals which were colocalized with Hoechst-stained nuclei as clear spots were counted manually in each field (410 x 330 mm). The number of average signals from 4 nonconsecutive sections per each animal was compared among experimental groups.2PO4, 2 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, 10 mM D-glucose). One microliter of CNQX (1 mg/ml in ACSF), or PPADS (1 mg/ml), or a mixture consisting of 0.5 μl of CNQX and 0.5 μl of PPADS was injected into the DVC  for 3 min by using a picoinjector . Histological examinations of brain sections by using hemotoxylin and eosin staining did not show any abnormalities in drug-treated animals compared to non-treated controls (data not shown). Drug injection was carried out 60 min after endotoxemia, and was immediately followed by AS.Mice were anesthetized with ketamine and xylazine. CNQX  and PPADS  were dissolved in artificial cerebrospinal fluid . The means of the data in individual groups were compared by using the Student’s t-test or the one-way or two-way ANOVA, with Tukey’s post-hoc tests for multiple comparisons (SPSS computer software version 12.0). Statistically significant differences were reported as *p < 0.05, **p < 0.01, or ***p < 0.001.To examine the regulatory effects of AS on serum TNF-α production, we applied MAC for 30 min on the ST36 acupoint 60 min after LPS, and determined serum TNF-α 30 min or 90 min after the termination of AS. We initially tried to inject with 60 μg/kg of LPS, a dose which has been used to induce TNF-α in rats . In our Macrophages in the spleen are the major source of inflammatory cytokines following LPS administration. Thus, we determined TNF-α production in the spleens of the animals given LPS and MAC, and investigated whether neural inputs to spleen were required for the regulation of TNF-α production. A splenic neurectomy abrogated efficiently the inhibition of TNF-α production in the serum by acupuncture when measured 30 min after AS. A vagotomy also elevated TNF-α levels, but less effective to eliminate the inhibitory effects of AS on TNF-α production . Then weNext, we examined c-Fos signals in the DVC area following LPS administration and AS. Previous studies have shown that TNF-α, which is peripherally produced after LPS administration, gains access to neurons in the DVC and induces c-Fos expression by modulating the neurotransmitter release from the vagal afferents . Here, oTo determine whether LPS or EAC can selectively modulate the activity of AMPA receptors and purinergic receptors, we analyzed c-Fos signals in cells positive to GluR2/3 receptors and P2X2 receptors in the DVC area. Some of GluR2/3-positive cells showed c-Fos signals in LPS-administered or EAC-treated animals arrow in . SimilarAnesthesia can attenuate TNF-α production in animals given LPS ,19. To eThe present study provides evidence that AS transmits signals into the vagus nerve and mediates anti-inflammatory responses in the spleen. Using an animal model of acute inflammation, we found that AS attenuated inflammatory responses as measured by TNF-α productions in the serum and the spleen. Removal of vagal and splenic nerves significantly abrogated the anti-inflammatory effects mediated by AS. Our study further shows that AS triggered neuronal activation in the DVC in which synaptic transmissions through both glutamate receptors and purinergic receptors are functionally involved.The vagus nerve is the major parasympathetic nerve that is responsible for physiological regulation of most internal organs. The afferent component in the vagus nerve transmits sensory information from the cardiopulmonary and gastrointestinal organs to the solitary nucleus to communicate with the efferent components in the DMV and the nucleus ambiguous that travel back to the target organs. Growing bodies of evidence indicate that vagus nerve activity is important not only for homeostatic regulation of internal organs but also for the regulation of pathologic inflammatory reactions; thus, the vagus nerve acts as a bridge between the neural and immune systems ,20. NotaHere, we found that TNF-α levels in the spleen as well as in the serum were decreased by AS in LPS-administered animals, but were elevated by a splenic neurectomy and a vagotomy. These results indicate that AS may activate the splenic nerve via vagus nerve activity to induce anti-inflammatory responses in the macrophages of the spleen. Having noted some previous reports indicating downregulation of TNF-α production by anesthetics ,23, we pWhile our data clearly showed downregulation of splenic TNF-α production by AS, one recent paper reported that decreases in serum TNF-α level after EAC at ST36 were not affected by a splenectomy . Here, tAnother issue that needs to be considered in relation to autonomic modulation of splenic TNF-α production is that the vagal branch to the spleen has not been clearly characterized so far –27, althAccording to our data, MAC, though its effect was milder than that of EAC, induced moderate levels of c-Fos expression in NTS and DMV neurons, indicating acupuncture-specific neuronal responses. As far as the sensory modalities, such as stretching, discriminative touch, and pricking, that can be elicited by AS are concerned , their n2+ levels [Then, how would AS signals be transmitted to DVC neurons and activate them? Glutamate is a primary neurotransmitter that mediates excitatory signals in neurons of the DVC including vagal afferent fibers, and an involvement of GABAergic inhibitory neurons in the DVC were previously described in relation to the vago-vagal gastric reflex . Interes+ levels . Astrocy+ levels . Given t+ levels ,38, it iGiven that ST36 is one of the most widely used acupoints for clinical therapy, acupuncture treatment at ST36 has been frequently investigated in animal experiments. Previous studies reported that acupuncture at ST36 was involved in regulating gastric motility and hyperalgesia associated with irritable bowel syndrome –41. Mole


Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.DOI:http://dx.doi.org/10.7554/eLife.03422.001 In response to an injury or an infection, areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body. At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apoptosis and necroptosis.Apoptosis is a highly controlled process during which the contents of the cell are safely destroyed in order to prevent damage to surrounding cells. Necroptosis, on the other hand, is not controlled: the cell bursts and releases its contents into the surroundings.Inflammation is activated by a protein called TNF, which is controlled by a complex that includes a protein called Sharpin. Mice that lack the Sharpin protein develop inflammation on the skin and other organs, even in the absence of injury or infection. However, it is not clear how the Sharpin protein controls TNF to prevent inflammation.Kumari et al. have found that inflammation in mice lacking Sharpin depends on TNF interacting with another protein called TRADD. The experiments also show that the inflammation is mainly driven by apoptosis, with necroptosis having only a minor role. Further experiments carried out in mammal cells showed that TRADD and another protein  work with Sharpin to prevent apoptosis.At the molecular level, Sharpin is known to induce a special type of protein modification  with two partner proteins, so the next challenge is to work out exactly how Sharpin uses this process to prevent apoptosis.DOI:http://dx.doi.org/10.7554/eLife.03422.002 Tumor necrosis factor receptor 1 (TNFR1)-mediated signaling is regulated by multiple ubiquitination events involving linear (Met1), Lys63 and Lys48 ubiquitination of several target proteins . Upon TNSharpincpdm/cpdm), which spontaneously develop severe chronic inflammation primarily in the skin but also in other tissues such as the gut, lung, liver, and esophagus  mice that lack TNFR1 specifically in keratinocytes   , we geneinocytes . These Sroscopic  or historoscopic  skin lesroscopic . Moreove compare . Taken tSharpincpdm/cpdm mice, we employed mice carrying conditional alleles for TRADD, an adapter molecule that is important for the induction of inflammatory and apoptotic signaling downstream of TNFR1   . Indeed,analysis . CollectSharpincpdm/cpdm mice develop splenomegaly and inflammation in other organs such as liver and lung   . We obsepdm mice , providiSharpincpdm/cpdm keratinocytes causes skin inflammation in Sharpincpdm/cpdm mice. We therefore investigated the molecular mechanisms by which Sharpin prevents TNFR1-induced apoptosis. We first examined the induction of apoptosis in MEFs derived from Sharpincpdm/cpdm mice stimulated with TNF alone or TNF + CHX using various assays. We observed increased sensitivity to TNF + CHX-induced apoptosis in Sharpincpdm/cpdm MEFs compared with wild type MEFs, as determined by immunocytochemical detection of cleaved caspase-3  inhibits TNF-induced apoptosis in keratinocytes. Ubiquitin signals have been implicated in the regulation of the death-inducing signaling complex (DISC). For example, cullin 3-based ubiquitination of caspase-8 by both Lys48- and Lys63-linked polyubiquitin chains brings caspase-8 into p62-containing aggregates leading to its activation and to commitment to apoptotic cell death . Furtherpe cells , it suggpe cells . Yet, Shpe cells . Interespe cells . This raSharpincpdm/cpdm C57BL/KaLawRij , pLKO.1-shRNA-mouse FADD (CCACACTTGGAGCCCAATAAACTCGAGTTTATTGGGCTCCAAGTGTGG), pLKO.1-shRNA-human HOIP (TGCTCCTTTGGCTTCATATATCTCGAGATATATGAAGCCAAAGGAGCA), pLKO.1-shRNA-human Sharpin (GTGTTCTCAGAGCTCGGTTTCCTCGAGGAAACCGAGCTCTGAGAACAC), pLKO.1-shRNA-human FADD (CATGGAACTCAGACGCATCTACTCGAGTAGATGCGTCTGAGTTCCATG), and pLKO.1-shRNA-human TRADD (CTGAAACTCCACTTGGCCTATCTCGAGATAGGCCAAGTGGAGTTTCAG) were generated by a standard subcloning method. The following antibodies were purchased and used according to the manufacturers’ recommendations: anti-cleaved caspase-3 antibody (Asp175) , anti-Vinculin antibody , anti-PARP antibody , anti-FADD antibody , anti-HOIP antibody , anti-TRADD antibody . Anti-Sharpin antibody has been described previously .2 condition in DMEM (Sigma) supplemented with 10% fetal calf serum  and 100 U/ml penicillin and streptomycin . Murine TNF was purchased from PeproTech .Human Embryonic Kidney (HEK) 293T cells , immortalized mouse embryonic fibroblasts (MEFs), and HaCaT cells (a kind gift from Máté Borsos) were maintained at 37°C in 5% COTissue samples from 12–18-week-old mice were fixed in 3.8–10% buffered formalin (skin) or 4% PFA  and subjected to histological analysis by H&E staining, TUNEL or immunohistochemical analysis. Slides were scanned using Mirax Slide Scanner . The following antibodies were used: K14, K6, K10 and Loricrin , F4/80 , active caspase-3 , and TRADD . Secondary antibodies were coupled to Biotin ; signal was amplified by avidin-biotin-HRP detection system  and detected by peroxidase substrate (VectorLab NovaRed). Sections were counterstained with hematoxylin for nuclei visualization. TUNEL staining was performed using TUNEL staining kit form Promega , according to the manufacturer’s instructions. Flow cytometric (FACS) analysis was performed on keratinocytes isolated from newborn pups as described previously  and incu6) were plated on 24-well plates. After 24 hr of subculturing, cells were treated with cycloheximide (CHX) (1 μM)  or TNF (10 ng/ml) (PeproTech) for the indicated times. Depending on the experimental set-ups, retroviral infection was combined. After the treatment, cells were harvested for SDS-PAGE followed by Western blot analysis. For the FACS analysis , the percentage of apoptotic cells was quantified by coupled annexin V antibody  staining and PI uptake. For WST-1 assay on MEFs, 9000 MEFs from wild type and TRADD-deficient mice were seeded in 96-well plates and treated with CHX (10 μg/ml), Necrostatin-1 (25 μM), and Z-VAD-FMK (20 μM) alone or in combination for 20 hr followed by incubation with WST-1 reagent  and measurement as per the manufacturer’s instructions. For WST-1 assay on keratinocytes, keratinocytes were isolated as described previously  followed by incubation with Alexa488 coupled anti-rabbit antibody (Invitrogen). Nuclei were stained by Vectashield mounting media with DAPI (VectorLabs).Lentiviral production and infection was performed according to the Addgene’s pLKO.1 protocol with a minor modification. Briefly, pLKO.1 vector with a packaging and envelope plasmids were transfected into HEK293T cells using Gene Juice (Novagen). After 36 hr of transfection, released lentivirus particles were filtered and used for infection of target MEFs or HaCaT cells using polybrene (4 μg/ml). After 48 hr of infection, cells were selected with puromycin (2 μg/ml). Expression of infected protein was monitored by western blotting.t test (two-tailed) by Prism 6 software (Graph Pad) or Microsoft Excel .Statistical significance was determined using ANOVA (one-way or two-way) and unpaired or paired Student's  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Sharpin prevents TNFR1-induced keratinocyte apoptosis and skin inflammation via linear ubiquitination of FADD” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.In this manuscript, Kumari et al. employ a variety of genetically modified mice and nicely demonstrate the FADD and TRADD-mediated TNF-induced apoptosis in epidermal keratinocytes is mainly responsible for skin inflammation and dermatitis in Sharpin-deficient cpdm mice. They also show that RIP3-mediated necroptosis only plays a minor role in the skin pathology, but the absence of TNFR1 and RIP3 can significantly (but not completely) block liver and lung inflammation as well as splenomegaly in Sharpin-deficient mice. Secondly, they show that Sharpin-deficient MEF cells are more sensitive to TNF induction of apoptosis and FADD appears to play an important role in this process. Thirdly, the rest of the manuscript tries to suggest that linear ubiquitination of FADD by Sharpin and LUBAC negatively regulates TNF-induced apoptosis, which may be the mechanism underlying the increase sensitivity of Sharpin deficiency to TNF-induced apoptosis. Overall, the first part of the story provides strong data and important insights into the mechanism of skin and multiple organ inflammation observed in cpdm mice. These, together with the second part of the story, nicely demonstrate the physiological function of Sharpin and likely also the LUBAC complex in negatively regulating apoptosis downstream of the death receptors. The third part aimed at providing a thorough biochemical mechanism for the apoptosis inhibition function of Sharpin is rather weak and in fact not essential for the story that is already pretty informative. Therefore, the authors should consider addressing the following comments when revising their manuscript.Major comments:1) For 2) The authors provide strong in vitro data that LUBAC can ubiquitinate FADD  and thatThe manuscript contains sufficient new information from 3) In view of the recent paper by Berger et al , cell death observed in the Sharpin deficient mice/cells could be expected to be RIPK1 kinase dependent. Therefore, the authors should use another cell model rather than the TNF/CHX treatment in MEFs, since this kills in a RIPK1 kinase-independent way and may not reflect the in vivo situation. In fact, keratinocytes are much more appropriate, for demonstrating that increased apoptosis in the skin is key to the development of skin inflammation and dermatitis in cpdm mice. The authors should examine whether TNF is sufficient to induce apoptosis in primary keratinocytes derived from cpdm mice and compare the cell death of WT keratinocytes. The authors then can further examine whether FADD and TRADD  deficiency can block the sensitivity of Sharpincpdm/cdpm keratinocytes. This can either be done by using the mice they already have or siRNA knockdown of FADD and TRADD. The authors argued that Sharpin prevents apoptosis independently from its role in NF-kB pathway and this should be further examined other relevant cellular systems in which the Sharpin deficient cells are killed by TNF without adding CHX.4) At the cellular level, Sharpin-deficiency sensitizes the cells to TNF-induced apoptosis. The authors should examine whether the apoptosis is RIPK1 kinase-dependent. They can also look at the RIPK1-containing complexes and compare with that in the presence of Sharpin.5) Sharpin is a component of the LUBAC complex. Another important question that the authors should address is whether Sharpin really functions through LUBAC in inhibiting TNF-induced apoptosis. Therefore, the authors should examine whether other LUBAC components are involved in the observed cellular apoptosis phenotypes, ideally in keratinocytes. 1) For, the authors should include littermate mice as the appropriate controls.Sharpincpdm/cpdm mice exhibited variability in the onset of the skin lesions, with some mice starting to show lesions as early as at 9 weeks of age and some as late as 12 weeks. Because of this variability in the onset of the phenotype even within the same litter, we have selected representative mice to show for each genotype that correspond to the average stage of disease observed among a large number of individual animals examined.Due to the complex breeding schemes required for the generation of some of the mouse lines described, it was not always possible to obtain littermates with all possible control genotypes. Whenever possible, we have made an effort to include littermate controls, but this was not always feasible. However, we would like to point out that the variability we observed in the phenotype of the cpdm mice was also seen within litters. Even in breeding of cpdm heterozygous mice without any additional alleles we often observed that littermate Specifically, we used the following controls for each of the lines:Sharpincpdm/cpdm/Tnfr1fl/fl/K14Cre line: For this line, we have used littermates that are cpdm/cpdm, Tnfr1fl/fl but not expressing the K14Cre transgene. Littermate control for this line is added in - Sharpincpdm/cpdm /Tnfr1-/- line: By breeding double heterozygous mice the probability of obtaining Sharpincpdm/cpdm/Tnfr1wt/wt or Sharpincpdm/cpdm/Tnfr1-/- mice is 1/16. By considering an average number of 6-7 pups per litter, the probability to have one Sharpincpdm/cpdm /Tnfr1wt/wt and one Sharpincpdm/cpdm/Tnfr1-/- mouse within the same litter is very low. In order to obtain the number of mice we needed for a comprehensive analysis of the phenotype we also used breeding of heterozygous cpdm mice that were homozygous Tnfr1-/- and compared the resulting mice to Sharpincpdm/cpdm or Sharpincpdm/cpdm/Tnfr1fl/fl mice from other breedings.- Sharpincpdm/cpdm /Faddfl/fl;Ripk3-/- line: For this line we compared littermates that are K14Cre positive (Sharpincpdm/cpdm/FADDE-KO/Ripk3-/-) that are FADD-deficient in keratinocytes with K14Cre negative (Sharpincpdm/cpdm/Faddfl/fl/Ripk3-/-) that express FADD in keratinocytes.- Sharpincpdm/cpdm /Faddfl/fl;Ripk3-/- line  in (left most panel), histology of liver and spleen has been replaced. In Therefore, we compared the effect of double FADD/RIPK3 knockout to that of single RIPK3 knockout. In addition, we have now added littermate controls for 2) The authors provide strong in vitro data that LUBAC can ubiquitinate FADD  and that Sharpin but not HOIL-1l/HOIP can bind to FADD directly. However, the functional data supporting that ubiquitination of FADD by LUBAC suppresses TNF-induced apoptosis is rather weak, indirect and not sufficiently convincing. The most important piece of data is the FADD K0 mutant rescue assay in, in which the difference of apoptosis induction (PARP and caspase-3 cleavage) between WT and FADD K0 complementation is very subtle and not sufficient to justify the conclusion.We agree with the reviewers and removed FADD-ubiquitination data, interaction assay of FADD and LUBAC components, and FADD K0 data and the related panels.The FADD-ubiquitin chimera experiment is difficult to interpret and should be removed from the manuscript, particularly in the absence of observing FADD ubiquitination in vivo.We agree with the reviewers and removed FADD-ubiquitin chimera data.The manuscript contains sufficient new information from; starting from characterizing the phenotype using various genetically modified mice to the demonstration that FADD is responsible for the increased apoptosis sensitivity to TNF stimulation. There is no need of defining the substrate of LUBAC involved in this process. If the authors really want to make the point here, they need to show in vivo linear ubiquitination of endogenous FADD upon TNF stimulation, ideally in keratinocytes, and this ubiquitination is absent in Sharpincpdm/cdpm cells.We thank the reviewers for this suggestion and reformatted the manuscript by focusing on the role of FADD in TNF-mediated apoptosis of sharpin-deficient cells without its ubiquitination. Therefore, panels which related to FADD ubiquitination, in which linear-ubiquitin chain interacter ABIN1 was tested were removed.3) In view of the recent paper by Berger et al , cell death observed in the Sharpin deficient mice/cells could be expected to be RIPK1 kinase dependent. Therefore, the authors should use another cell model rather than the TNF/CHX treatment in MEFs, since this kills in a RIPK1 kinase-independent way and may not reflect the in vivo situation. In fact, keratinocytes are much more appropriate, for demonstrating that increased apoptosis in the skin is key to the development of skin inflammation and dermatitis in cpdm mice. The authors should examine whether TNF is sufficient to induce apoptosis in primary keratinocytes derived from cpdm mice and compare the cell death of WT keratinocytes.in vivo studies. To assess TNF-induced death we prepared primary keratinocytes lacking sharpin, sharpin and RIPK3, sharpin and TNFR1 and sharpin, RIPK3 and FADD as well as control cells from littermates as shown in Sharpincpdm/cpdm keratinocytes by about 8-10% compared to heterozygous or wild type (Sharpincpdm/wt or Sharpinwt/wt ) control kearatinocytes. This effect of TNF was too weak to provide robust data allowing the comparison between the different genotypes, as a reduction of viability by 8-10% is within the range of the normal variability observed in this assay using primary keratinocytes. We conclude that while TNF clearly induces apoptosis of sharpin-deficient keratinocytes in vivo that is responsible for skin inflammation in cpdm mice, in primary keratinocyte cultures TNF stimulation does not induce considerable amount of cell death in the absence of CHX to allow reliable quantification of the effect. It is possible that the in vivo context and the keratinocyte differentiation status are responsible for the difference between the epidermal keratinocytes in vivo and the primary cultured cells.We agree with the reviewers that addressing the role of Sharpin in TNF-induced death of keratinocytes is more relevant to the in vivo result from Berger et al. (2014).To bypass this problem, in parallel we performed experiments in a human keratinocyte cell line, HaCaT cells, in the presence or absence of Sharpin by knockdown. We observed that TNF alone induced mildly elevated apoptosis in Sharpin-knockdown HaCaT cells compared to control cells. Although the increase in cell death was in the range observed for the primary cells, we were able to quantify this difference reliable using Annexin V staining and measurement of caspase-8 activity new . These rThe authors then can further examine whether FADD and TRADD  deficiency can block the sensitivity of Sharpincpdm/cdpm keratinocytes. This can either be done by using the mice they already have or siRNA knockdown of FADD and TRADD. The authors argued that Sharpin prevents apoptosis independently from its role in NF-kB pathway and this should be further examined other relevant cellular systems in which the Sharpin deficient cells are killed by TNF without adding CHX.To examine whether FADD and TRADD deficiency can block the TNF-induced sensitivity of Sharpin-deficient HaCaT cells, we generated knockdown HaCaT cells lacking Sharpin+FADD or Sharpin+TRADD and examined TNF -induced apoptosis with and without CHX new . We obseSharpincpdm/cpdmor Sharpincpdm/wt;Faddfl/fl;Ripk3-/- lines, stimulated with TNF alone or with TNF + CHX and analyzed their viability using the WST-1 assay. Interestingly, the increased sensitivity of Sharpincpdm/cpdm keratinocytes to TNF+CHX was fully rescued in Sharpincpdm/cpdm;FADDE-KO;Ripk3-/ keratinocytes. However keratinocytes obtained from Sharpincpdm/cpdm;Faddfl/fl;Ripk3-/- mice showed similar response to TNF+CHX as observed in Sharpincpdm/cpdm keratinocytes  At the cellular level, Sharpin-deficiency sensitizes the cells to TNF-induced apoptosis. The authors should examine whether the apoptosis is RIPK1 kinase-dependent. They can also look at the RIPK1-containing complexes and compare with that in the presence of Sharpin.As we mentioned above (#3), based on the effect of RIPK1 inhibitor Nec-1 on TNF-treated Sharpin deficient HaCaT cells, RIPK1 is suggested to be involved in Sharpin-dependent apoptosis regulation in keratinocytes. We agree that to understand the RIPK1 complex with and without Sharpin in keratinocytes is important and interesting. Unfortunately, we could not observe the RIP1-containing complex with LUBAC components by performing co-IP using anti-RIP1 antibody, with TNF+zVAD-treated HaCaT cells, leaving out the question whether Sharpin affects the RIP1-complex formation.5) Sharpin is a component of the LUBAC complex. Another important question that the authors should address is whether Sharpin really functions through LUBAC in inhibiting TNF-induced apoptosis. Therefore, the authors should examine whether other LUBAC components are involved in the observed cellular apoptosis phenotypes, ideally in keratinocytes.We agree that it is an important point. To address this issue, we generated HOIP-knockdown HaCaT keratinocyte cell line using shRNA. TNF alone or TNF+CHX induced increased apoptosis of HaCaT cells (lacking HOIP), suggesting an important role of the LUBAC complex in the preventing TNF induced apoptosis in keratinocytes. New data are added in


E. coli, the DNA helicase is DnaB and DnaC is its loading partner. We used the method of hydrogen/deuterium exchange mass spectrometry to address the importance of DnaB–DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring. This event correlates with conformational changes of the RecA fold of DnaB that is involved in nucleotide binding, and of the AAA+ domain of DnaC. DnaC also causes an alteration of the helical hairpins in the N-terminal domain of DnaB, presumably occluding this region from interacting with primase. Hence, DnaC controls the access of DnaB by primase.Helicase loading at a DNA replication origin often requires the dynamic interactions between the DNA helicase and an accessory protein. In  E. coli to form primers, it must interact with the DNA helicase named DnaB bound to the parental DNA strand that serves as the lagging strand template (E. coli replication origin (oriC) . These template ,3,4. Afttemplate . Primasen (oriC) . Hence, 4, an analogue of GTP, is an open ring analogous to a lock washer with a right-handed turn in which the DNA is in the interior channel , and requires that the helicase is complexed to DnaC in which a maximum of six DnaC monomers are bound  analysis of the BC complex  and fluohelicase , shows thelicase . Reconst of DnaB . This mo of DnaB , suggest2O (99.9 atom% D) and ATP were purchased from Sigma–Aldrich Chemical Co. ATPγS and Poros-20AL were from EMD-Millipore and Lifetechnologies, respectively. All other chemicals were HPLC grade. DnaB and DnaC were purified from overproducing strains essentially as described  glycerol, 20 mM NaCl, 2 mM DTT) and 2 mM ATP or 10 μM ATPγS followed by isolation on a precalibrated Superose 12 column equilibrated in the respective buffer. Quantitative densitometry of peak fractions visualized in a Coomassie Blue-stained SDS-polyacrylamide gel confirmed the composition of the DnaB6–DnaC6 complex. Given the binding affinities of DnaB  and Dnaith ATP; –27), them/z 50–2000 at a mass resolution (M/ΔM) of approximately 20 000 . Data analysis was performed using HX Express software and MS tools (A column (2 mm x 50 mm) containing pepsin immobilized on Poros-20AL resin was prepared essentially as described . After iMS tools ,38.2O, and incubated for 24 h at 20°C before the addition of formic acid, online pepsin digestion and analysis by mass spectrometry. To measure H/D exchange at various time intervals, DnaB, DnaC or the BC complex  in 10 μl of Buffer A supplemented with ATP or ATPγS was diluted with this buffer but made with D2O. At the indicated times, the reactions were placed on ice, and H/D exchange was quenched by promptly adding an equal volume of chilled 1% formic acid. Samples were then immediately injected onto the pepsin column followed by mass spectrometry analysis as described above. To determine the reproducibility of measurements, at least three independent sets of experiments with ATP or two with ATPγS were performed from which the standard deviation or range was calculated.As a control and also to determine the identities of undeuterated peptides, DnaB or DnaC  in 10 μl of Buffer A supplemented with 2 mM ATP was diluted 10-fold at 20°C with this buffer. After the addition of an equal volume of ice-cold 1% formic acid, online pepsin digestion and analysis were performed in MS/MS mode as described above. Using Biolynx software (Waters), the sequence information obtained from the analysis of peptides was compared with the amino acid sequence of DnaB and DnaC. As another control to measure the level of back-exchange during pepsin treatment, the fully deuterated proteins were analyzed. Depending on the individual peptide, back-exchange was from 30–50%, which is similar to studies by others . Brieflypd−Mnd)/N) where Mpd is the mass of the partially deuterated peptide, Mnd is the mass of the non-deuterated peptide, and N is the total number of exchangeable amide hydrogens within the peptide.Deuteration percentages were calculated using the following formula:% D = 100. As the peptides were compared under essentially identical conditions, the results were not corrected for back exchange, which was from 30–50% depending on the individual peptide. The overall sequence coverage of DnaB and DnaC peptides identified was 87% and 90%, respectively , peptides in the first group displayed fast exchange kinetics even at early time points . For other peptides, their slower deuterium incorporation that progressed up to 30 min may be explained by amide hydrogen bonding with other segments of DnaB that leads to intermediate exchange rates. The third group contains peptides that are at or near the dimer interface of DnaB, and exhibited either much slower or marginal exchange rates that indicate a more rigid local structure and strong hydrogen bonding. By comparison, peptides including one (267–281), which contains part of the hexamerization pocket . Together, these observations support the validity of the homology model.The HDX results obtained for peptides of DnaB served as the control for the analysis of exchange rates for the BC complex, which are described later. We grouped the DnaB peptides by their rates of hydrogen/deuterium exchange. Consistent with their locations on the surface of a homology model of one 267–21, which We then compared the HDX kinetics of peptides of DnaB when complexed with DnaC with that of DnaB alone Figure . This anE. coli DnaB, residues 182–198 of E. coli DnaB function as the linker helix, making intimate contact with residues in the hexamerization pocket (amino acids 264–308) in the CTD of the neighboring DnaB protomer . Compared with others, the alleles encoding I297A, L304A and E435A substitutions were defective in complementing the dnaB mutant at non-permissive temperature under conditions in which the dnaB+ plasmid was active. We also used an ELISA assay that measures the direct interaction of DnaB with DnaC, and showed that the E435A substitution blocks this interaction and also inactivates DnaB function in DNA replication of an oriC-containing plasmid whereas other substitutions  do not . Supported by observations described below, our results identify critical DnaB residues that DnaC recognizes to form the BC complex.An objective was to understand how DnaC interacts with DnaB to form the BC complex. In the absence of DnaC, the moderate exchange rate of DnaB peptides containing amino acids 295–304 and 431–435 contrasts with their greatly reduced rates when DnaC was bound Figure . These rBy velocity sedimentation analysis in buffer containing magnesium ion and either ATP or ADP, DnaC is a monomer ,49. Its E. coli DnaB and DnaC were prepared using the X-ray structures of G. stearothermophilus DnaB (PDB code 2R6A) and the AAA+ domain of G. kaustophilus DnaI (PDB code 2W58) as templates (A. aeolicus DnaA (PDB code 2HCB), a DnaC paralog  mutant by the I297A and L304A substitutions. Taken together, these observations validate the model.With either adenine nucleotide, DnaC peptides bearing amino acids 8–11 and 31–44 displayed greatly reduced exchange kinetics when complexed with DnaB Figure . These remplates . DnaB wa paralog . Becausees 1–64; , PSIPREDes 1–64; . The NTDE. coli carrying a K112R substitution of the conserved Walker A lysine (Lys112 in GKPGTGKN from residues 106–113) is deficient in both ATP binding and helicase loading at oriC  and an adjacent segment  was inhibited when DnaC was complexed to DnaB Figure ,27.E. coli DnaC has two conserved arginines (Arg216 and Arg220) that are essential  in DnaC interact with a site (residues 431–435) in DnaB. That Glu435 of DnaB interacts with DnaC supports this conclusion . Of intE. coli DnaB). The linker helix peptide (187–196) displays bimodal exchange kinetics, reflecting opening of the DnaB ring. We expected but failed to detect a similar exchange pattern for peptides 267–281, and 277–289 that compose the hexamerization pocket. Perhaps ring opening causes only a few amide hydrogens in these peptides to exchange, which is difficult to detect (X-ray structures show that the hexamerization pocket, a hydrophobic surface formed by three α-helices, cradles the linker helix of the adjacent protomer in the DnaB hexamer (residues 264–308 and 182–198, respectively, for o detect ,67. A pa


Currently, no satisfactory treatment is available for sciatica caused by herniated discs and/or spinal stenosis. The objective of this study is to assess the value of tumor necrosis factor (TNF)-α inhibitors in the treatment of sciatica.Without language restrictions, we searched PubMed, OVID, EMBASE, the Web of Science, the Clinical Trials Registers, the Cochrane Central Register of Controlled Trials and the China Academic Library and Information System. We then performed a systematic review and meta-analysis on the enrolled trials that met the inclusion criteria.Nine prospective randomized controlled trials (RCTs) and two before-after controlled trials involving 531 patients met our inclusion criteria and were included in this study. Our systematic assessment and meta-analysis demonstrated that in terms of the natural course of the disease, compared with the control condition, TNF-α inhibitors neither significantly relieved lower back and leg pain (both p>0.05) nor enhanced the proportion of patients who felt overall satisfaction ) or were able to return to work (return to work)  at the short-term, medium-term and long-term follow-ups. In addition, compared with the control condition, TNF-α inhibitors could reduce the risk ratio (RR) of discectomy or radicular block  at medium-term follow-up, but did not decrease RR at the short-term  and long-term follow-ups .The currently available evidence demonstrated that other than reducing the RR of discectomy or radicular block (combined endpoint) at medium-term follow-up, TNF-α inhibitors showed limited clinical value in the treatment of sciatica caused by herniated discs and/or spinal stenosis. Disk herniation-induced sciatica is one of the most common causes of lower back and leg pain among young adults. Previous studies have demonstrated that the outcomes of conservative treatment, such as medication and physical therapy, are similar to the natural course of this disease Tumor necrosis factor-alpha (TNF-α) is an inflammatory factor involved in the pathophysiological mechanism underlying disk herniation-induced sciatica The primary purpose of this study was to evaluate the treatment value of TNF-α inhibitors compared with placebos and steroids in terms of five endpoints at short-term follow-up (≤3 months), medium-term follow-up (3 to 12 months) and long-term follow-up (≥12 months). The five endpoints that were adopted were the Oswestry Disability Index, VAS pain intensity in the leg, VAS pain intensity in the lower back, global perceived effect (satisfaction) or return to work (combined endpoint), and discectomy or radicular block (combined endpoint). The secondary purpose was to evaluate the patient tolerance of the adverse reaction of TNF-α inhibitors.et al.Using the “Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA)” The searched database included the following: PubMed, OVID, EMBASE, the Web of Science, the Clinical Trials Registers, the Cochrane Central Register of Controlled Trials and the China Academic Library and Information System. The search terms included following: “anti-tumor necrosis factor agents OR tumor necrosis factor alpha inhibitor OR infliximab OR adalimumab OR etanercept OR rituximab OR golimumab OR certolizumab OR efalizumab OR ustekinumab OR alefacept” AND “sciatica OR lumbosacral radiculopathy” AND “controlled trial” appearing in “title/abstract”. Each database was searched from January 1, 2000 to July 1, 2013. No language restrictions were applied.The inclusion criteria were as follows: (1) Participants: all patients included were older than 18 years and were diagnosed with sciatica caused by lumbar disc herniation and/or lumbar spinal stenosis confirmed with CT/MRI, regardless of the duration of symptoms. Patients who planned to undergo discectomy soon or had comorbid liver disease, tuberculosis, spinal cord tumor, infection or trauma were excluded; (2) Intervention: any trial that used TNF-α inhibitors in the TNF-α inhibitor group and placebos or steroids in the control group and in which all drugs were locally injected or systematically administered; (3) Endpoints: any trial that used subjective parameters, such as the Oswestry Disability Index and VAS scores, to evaluate lower back and leg pain and used global perceived effect (satisfaction) or return to work (combined endpoint) to represent the proportion of patients who felt overall satisfaction or were able to return to work, and adopted an objective parameter, discectomy or radicular block (combined endpoint), to evaluate the risk ratio (RR) of discectomy or radicular block; (4) Study type: the controlled trials including randomized controlled trials (RCTs), cross-over controlled trial, non-randomized concurrent trials, before-after controlled trial, and case-control study, were included regardless of their sample size and trial results.et al.Using a unified form, two investigators extracted the data and established the data spreadsheet independently. Finally, they confirmed the accuracy of the data together, and discrepancies were resolved via discussion until a consensus was reached. A portion of the endpoint data expressed only as a line graph or histogram was obtained from the forest plots of the study conducted by Williams One investigator performed a methodology quality assessment of all included studies based on a 17-item quality evaluating system Using WMD, standardized mean difference (SMD) and RR, we performed a systematic review and meta-analysis of the aforementioned five endpoints according to the follow-up time and the type of control drugs used. For global perceived effect (satisfaction) or return to work (combined endpoint), an RR>1 indicated that the outcomes of the TNF-α inhibitor group were superior to those of the control group; for discectomy or radicular block (combined endpoint), an RR<1 indicated that the outcomes of the TNF-α inhibitor group were superior to those of the control group; for Oswestry Disability Index, VAS-leg, and VAS-lower back, a negative WMD or SMD indicated that the outcomes of the TNF-α inhibitor group were superior to those of the control group. The data from reports concerning same trial were used for the analysis of the corresponding follow-up. Prior to the meta-analysis, for each endpoint, Cochran’s Q statistic test was applied to assess the heterogeneity among the included studies. If a p-value of Cochran’s Q statistic (Qp) ≥0.10, which indicated the absence of heterogeneity, a fixed-effects model was applied; otherwise, a random effects model was applied for analysis. Stata statistical software version SE 12.0  was utilized for all statistical analysis.A total of 113 records were identified through database searches, and 16 remained et al.et al.et al.The trial by Cohen The results derived from the meta-analysis showed that compared with the placebo group, the TNF-α inhibitor group had a WMD of −5.34  at the post-injection short-term follow-up, −8.19  at the medium-term follow-up  at the post-injection short-term follow-up, −0.24  at the medium-term follow-up, and 0.03  at the long-term follow-up; compared with the steroid group, the TNF-α inhibitor group had an SMD of −1.22  at the post-injection short-term follow-up .et al.et al.A trial by Cohen et al.et al.The trial by Genevay The results derived from the meta-analysis indicated that compared with the control group, the TNF-α inhibitor group had an RR of 0.64  for the discectomy or radicular block (combined endpoint) at the post-injection short-term follow-up, 0.51  at the medium-term follow-up and 0.64  at the long-term follow-up . The senFour reports indicated the absence of adverse reaction The major findings of this study were that TNF-α inhibitors could reduce the RR of discectomy or radicular block (combined endpoint) at medium-term follow-up, but not at short-term and long-term follow-ups; during the natural disease course, TNF-α inhibitors neither provided additional pain relief nor improved the percentage of global perceived effect (satisfaction) or return to work (combined endpoint); the observed adverse reaction were mild and could not be proven to have any association with the use of TNF-α inhibitors.Previous studies have found that in patients with intervertebral disc herniation, the TNF-α level on the articular surface increased et al.et al.et al.et al.Our explanations regarding the finding that TNF-α inhibitors could reduce the risk of discectomy or radicular block (combined endpoint) but could not improve the pain were as follows: (1) TNF-α inhibitors reduce the risk of discectomy or radicular block (combined endpoint) because of their neuroprotective function. Genevay et al.Interestingly, we found that TNF-α inhibitors could reduce the RR of discectomy or radicular block (combined endpoint) at medium-term follow-up, but not at short-term and long-term follow-up. Regarding this phenomenon, our explanations are as follows: (1) the neuroprotective role of TNF-α inhibitors cannot been fulfilled within a short period of time; (2) we conservatively speculated that TNF-α-related inflammation and neurotoxicity are not the major pathophysiological mechanisms of sciatica; thus, TNF-α inhibitors might not exhibit clinical value at long-term follow-up; (3) the bias of drug administration approaches might be involved in this result. Although the currently available evidence is not sufficient to determine the superiority of local injection and systematic drug administration, the efficacy of intravenous injection is dubious. The sensitivity analysis demonstrated that after the exclusion of three trials in which the drugs were administered through intravenous injection Regarding the finding that TNF-α inhibitors reduced the risk of discectomy or radicular block (combined endpoint) but did not increase the percentage of global perceived effect (satisfaction) or return to work (combined endpoint), we believe a possible explaination could be as following: the endpoint, global perceived effect (satisfaction) or return to work (combined endpoint), is related to the patients’ self-perceptions (such as pain), while discectomy or radicular block (combined endpoint) is related more closely to the patients’ functional status, particularly in the case of discectomy.It is worth noting that in this study, we could not draw a definitive conclusion regarding the appropriate selection of the drug dose and the frequency of drug administration based on the current available evidence. Because the endpoint data of both the treatment and control groups changed proportionately (10 times), the differences in the VAS score ranges among the enrolled trials would not affect the quantitative analysis and result interpretation in the meta-analysis using SMD.et al.Compared with the study by Williams et al.The limitations of this study are as follows: (1) the sample size was small, and the follow-up durations were inconsistent; (2) the data showed a skewed distribution, and data expressed with the mean value and without a standard deviation  at the medium-term follow-up, TNF-α inhibitors have limited clinical value in the treatment of sciatica caused by disc herniation and/or spinal stenosis.


One possible way of measuring the broad impact of research  quantitatively is the use of alternative metrics . An important source of altmetrics is Twitter, which is a popular microblogging service. In bibliometrics, it is standard to normalize citations for cross-field comparisons. This study deals with the normalization of Twitter counts (TC). The problem with Twitter data is that many papers receive zero tweets or only one tweet. In order to restrict the impact analysis on only those journals producing a considerable Twitter impact, we defined the Twitter Index (TI) containing journals with at least 80 % of the papers with at least 1 tweet each. For all papers in each TI journal, we calculated normalized Twitter percentiles (TP) which range from 0 (no impact) to 100 (highest impact). Thus, the highest impact accounts for the paper with the most tweets compared to the other papers in the journal. TP are proposed to be used for cross-field comparisons. We studied the field-independency of TP in comparison with TC. The results point out that the TP can validly be used particularly in biomedical and health sciences, life and earth sciences, mathematics and computer science, as well as physical sciences and engineering. In a first application of TP, we calculated percentiles for countries. The results show that Denmark, Finland, and Norway are the countries with the most tweeted papers (measured by TP). The success of the modern science system is closely related to a functioning research evaluation system by peers: without critical assessments by peers improvements of research approaches would be absent and standards could not be reached Bornmann . With thOne possible way of measuring broad impact quantitatively is the use of alternative metrics  Bornmann —a new suIn bibliometrics, it is standard to normalize citations  in a feed. Also, one can “retweet” these messages or tweet new short messages which are read by own followers in their feeds. Up to a third of the tweets may be simple retweets Holmberg . “TweetsIt is possible to include references to publications in tweets: “We defined Twitter citations as direct or indirect links from a tweet to a peer-reviewed scholarly article online” (Priem and Costello tweeted = 3.3). Of the 284,764 research articles and reviews assigned to this discipline, 27,878 were mentioned on Twitter a total of 90,633 times. Twitter coverage is lowest for Physics papers covered by PubMed (1.8 %), and Mathematics papers related to biomedical research receive the lowest average number of tweets per tweeted document (T/Ptweeted = 1.5)” (p. 662). According to Zahedi, Costas, and Wouters  from AltmetricWe obtained the Twitter statistics for articles and reviews published in 2012 and having a DOI  and pools the journals with the most Twitter activities in the so called Twitter Index (TI).i is as followsi is most frequently read. “Saves” at Mendeley are interpreted in altmetrics as “reads” and Mendeley readers share their discipline. (2) All papers which are assigned to the same discipline in Mendeley and are published in the same year (these papers constitute the reference set of paper i) are sorted in descending order according to their TC. (3) The proportion of papers is determined in the reference set which received less tweets than paper i. (4) The proportion equals the percentile for paper i.Following percentile definitions of Leydesdorff and Bornmann , ImpactSIt is a sign of professional scientometrics to use normalized indicators. Compared to other methods used for normalization in bibliometrics, percentile-based indicators are being seen as robust indicators  and 9242 journals with TC for less than 80 % (95.7 %). We propose to name the set of journals with high Twitter activity as TI. Because many TI journals have published only a low numbers of papers, we reduced the journals in the TI further on (this will be explained later on in this section). We propose to compose the TI every 12 months (e.g. by Twitter). In other words, every 12 months the journals should be selected in which at least 80 % of the papers had at least one tweet. Then, the papers in these journals are used for evaluative Twitter studies on research units (e.g. institutions or countries).i − 0.5)/n*100) is used very frequently nowadays for the calculation of percentiles  and 50 % of the values have a lower rank (fewer tweets). Papers with equal TC are assigned the average rank i in the table. For example, as there are two papers with 44 tweets, they are assigned the rank 9.5 instead of the ranks 9 and 10.In order to normalize tweets, we propose to calculate percentiles (following ImpactStory) on the base of the tweets for every paper in a journal. There are several possibilities to calculate percentiles (it is not completely clear which possibility is used by ImpactStory). The formula derived by Hazen  , In the second step of the test, all papers are grouped by the main disciplines as defined in the OECD field classification scheme.for each OECD field—using the binary variable from the first step. If TP were fair, the proportions within the fields should equal the expected value of 10 %. Furthermore, TC should show more and greater deviations from 10 % than TP.In the third step of the test, the proportion of papers belonging to the 10 % most frequently tweeted papers is calculated The results of the fairness tests are shown in Table The largest deviations of the proportions from 10 % can be expected for TC, because TC is not field-normalized. In the interpretation of the proportions in Table The TP shows the intended results in Table The results of the comparison between TC and TP based on ACCS are shown in Table Taken as a whole, the proportions for TC in Tables In the final part of this study, we use TP to rank the Twitter performance of countries in a first application of the new indicator. The analysis is based on all papers (from 2012) published by the countries which are considered in the TI. Since the results in the section “rs = 0.9. Thus, the difference in both indicators to measure Twitter impact on the country level is small.The Twitter impact for the countries is shown in Table While bibliometrics is widespread used to evaluate the performance of different entities in science, altmetrics offer a new form of impact measurement “whose meaning is barely understood” yet . The high correlation coefficient points out that there are scarcely differences between the indicators to measure Twitter impact. The high correlation might be due to the fact that most of the papers used belong to only two fields  whereby the variance according to the fields is reduced between the papers.We used the fairness test in order to study the field-independency of TP (in comparison with TC). Whereas one test based on the OECD fields shows favorable results for TP in all fields, the other test based on an ACCS points out that the TP can be validly used particularly in biomedical and health sciences, life and earth sciences, mathematics and computer science, as well as physical sciences and engineering. In a first application of TP, we calculated percentiles for countries whereby this analysis show that TP and TC are correlated on a much larger than typical level (This paper proposes a first attempt to normalize TC. Whereas Mendeley counts can be normalized in a similar manner as citation counts (Haunschild and Bornmann Besides further studies which address the normalization of TC and refine our attempt of normalization, we need studies which deal with the meaning of tweets. Up to now it is not clear what tweets really measure. Therefore, de Winter  speculat


Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3′ that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports. Membrane fusion between HIV and host cells is mediated by the Env complex on the surface of the viral membrane envelopePreviously, we showed that the structure of gp140ΔV2TV145781011131415161718Our current results challenge the prevailing perspective, and posit that instead of being situated at the trimeric apex, the V2 loop is situated at the basal region of the trimer, oriented toward the adjacent gp120 subunit and associating with that subunit's V3 loop, forming the quaternary neutralizing epitope (QNE). Indeed, exactly how juxtaposed V2 and V3 loops from the same subunit, a tertiary epitope, would form the basis for the QNE, preferentially recognized on trimeric Env823256272930632333435Biochemical and structural characterization of the recombinant, soluble trimeric immunogen gp14035348Attempts at elucidating the structure of the Env complex to understand its mechanism of action have yielded disparate results with no clear consensus841424344418431016141517Here we present the structures of a clade C trimeric, full-length soluble gp140 immunogen from the TV1 strain in the absence and presence of the CD4-mimetic miniprotein CD4mz axis; the gp140ΔV2 construct shows gp120 subunits at a tilt of approximately 25°, whereas the gp140 construct exhibits a tilt of about 15°–20°. A more relevant feature is the presence of a tail-like density at the putative location of the V2 loop , all normal to the threefold z axis, then each subunit would appear tilted away from the z axis about the Sx axis by ~25°, similar to the unliganded gp140ΔV2 structure. The three gp120 regions appear to be more closely associated near the trimer apex, with density at the outermost tip of each gp120 subunit oriented toward the threefold axis. The wedge-shaped trimer arm region is quite consistent when compared to the gp140ΔV2 map.Isosurface rendering of the gp140TV1 (hereinafter gp140) density map showed that the trimer structure retained the propeller-shaped motif that gp140ΔV2 exhibited . The thr V2 loop . Two disComparative analysis of both maps places the location of V2 on the outer edge of each trimer fan blade proximal to the viral membrane, supporting our earlier docking of coordinates in the gp140ΔV2 density map . The V1 Conjugation of gold-labeled PG16 Fab′ fragments with trimeric gp140 followed by single particle reconstruction of gp140-PG16 complexes revealed a density protruding from the basal V2 location . The gp1In order to determine the relative accessibility of apical or basal gp41 epitopes as a function of V2 deletion, we probed full-length and ΔV2 gp140 constructs with anti-gp41 antibodies targeting apical  and basal  epitopes by western blot. There were no differences in 50–69 epitope accessibility between full-length and truncated gp140, while there was a marked difference in accessibility to 2F5 epitope, with full-length gp140 shielding the epitope and gp140ΔV2 displaying increased access to it . Epitopez-axis  were docked into the density map, resulting in good agreement (correlation coefficient of 0.79) . The coo3, 15870 Å3, and 12250 Å3, respectively. The density map of gp140 reaffirms our previous findings suggesting that the CD4 binding loop (encompassing primarily residues 368–370) is perpendicularly exposed and thus available for receptor binding , close enough to ostensibly be accessible by one Fab fragment. Such a quaternary epitope would constitute increased intersubunit gp120-gp120 interactions when compared to the gp140ΔV2 construct, resulting in the decreased trimer radius. The V4 loop appears to be situated near the trimer apex , with it binding .To determine the rigidity of variable loops, we computed 2D histograms based on gradient intensity of voxels, so as to visualize structurally stable core regions of the trimer as well as more structurally labile regions. Taking the derivate of intensities can augment detection of the solvent-protein interface, as well as differentiate between structurally rigid and labile regions. Taking regions from the 2D histogram, corresponding to a fixed contour at different gradient intensity values, revealed distinct density profiles not observed by traditional 1D volume histogram cutoffs. The gp140 unliganded, 2D-segmented map revealed that the protruding basal regions as well as the apical region were present at low gradient intensities but not at higher gradient intensities , revealiThe location of the V2 loop presented herein, substantiating our previous work, suggests that V2 indirectly occludes the CD4BS through steric clashes of V4 glycans at the trimeric apex, near the three binding sites for CD4, through intersubunit association with the neighboring subunit's V3′ loop. Biochemical evidence of gp41 epitope accessibility upon V2 deletion corroborates our finding that while apical gp41 exposure is unaffected, V2 deletion results in exposure of basal gp41 epitopes targeted by mAb 2F5.The juxtaposition of V2 and V3′ loops from different subunits explains why partial deletion of V2 confers enhanced CD4 binding capability563325The current density map, with the wedge-like gp120 subunit densities amenable to docking of unliganded coordinates, again corroborates our previous docking orientation, such that CD4 approaches the trimer at an angle of approximately 60°–90° from the horizontal plane . The expA recent report on Moloney murine leukemia virus, another retrovirus, indicates that Env undergoes subunit rearrangement following furin cleavageThe V3′ loop protrudes laterally toward the adjacent clockwise gp120 subunit and V2 loop, thus providing a footprint for QNE-recognizing antibodies such as PG16 and 2909243The locations of V2 and V3′ on gp120 in the trimeric context appear to preclude the possibility of their joint accessibility on a single gp140 protomer, where the tips of V2 and V3 are found upwards of 60 Å apart, as observed from PDB coordinates 2B4CThe V3 loop is occluded in the trimeric form of EnvThe location of V4 herein presents an alternative to current views that V2 dominates the apical trimer region. Glycosylated asparagines in V4 could provide the immunologically silent faceA large site of immune evasion at the V4 and V5 locations has been identifiedIn contrast to earlier tomographic Env structures, recent structures of clade A and B gp14020The ability of oligomeric gp140 to elicit NAbs with potency and breadth, outlining its feasibility as a vaccine candidate, has been a point of debate. Clade B gp140SF162 immunogens with a 30-residue deletion in the V2 loopWe present the structure of a full-length, clade C trimeric gp140 immunogen, and compared this structure to our previous structure of a clade C gp140 trimer with a partial V2 deletion24Galanthus nivalis agarose lectin column, a diethylaminoethyl column, and a hydroxyapatite column, sequentially, in order to achieve high purity (>95%). Methods to image soluble gp140 trimers embedded in vitreous ice using cryoelectron microscopy have also been described previouslyMethods to purify gp140 have been described elsewhereGalanthus nivalis agarose (GNA) lectin column was used to capture the glycosylated Env construct, followed by a diethylaminoethyl (DEAE) column to capture acidic proteins and nucleic acids. A ceramic hydroxyapatite (CHAP) column was used to capture contaminating proteins while Env flowed through, and size exclusion chromatography isolated the trimeric form. Samples were prepared on holey carbon grids as previously described3The characterization and purification of subtype C, full-length oligomeric gp140 was performed in the same manner as gp140ΔV232 fragments, reduced using 50 mM Tris(2-carboxyethyl) phosphine hydrochloride  for 10 min to yield reduced Fab′ fragments, and conjugated to gold particles for 30 mins, following manufacturer's protocol. The Fab′-gold conjugates were incubated with gp140 at a molar ratio of 3:1 at 4°C overnight.The antibody PG16, preferentially targeting V2 but also V3 to a lesser extent, was provided by the NIH AIDS Reagent Program. PG16 was digested using a Pierce Fab′ digestion kit  and conjugated to 1.4 nm gold fragments Nanogold® . PG16 was digested to F(ab′)−/Å2. For the native state, 113 micrographs and 64 micrographs for the CD4m-bound state were recorded at 80,000× magnification  correction was performed using Western blots were performed by incubating gp140 and gp140ΔV2 at 0.3 mg/ml with 50–69 and 2F5 antibodies (NIH AIDS Reagent Program) at 2 μg/ml, transferring to PVDF membrane overnight, blocking with 5% milk for 3 h, then washing in 0.1 M PBS-T 3X. The gp140-2F5, gp140ΔV2-2F5, gp140-50-69, and gp140ΔV2-50-69 complexes were then incubated in anti-human alkaline phosphatase-conjugated secondary antibody  diluted 1:500 for 1 h, washed in 0.1 M PBS-T, and the membranes were developed in SIGMAFAST™ BCIP®/NBT .ELISAs were conducted on 96-well plates by adsorbing gp140 at 0.3 mg/ml, incubating with 50–69 and 2F5 at 2 μg/ml, applying anti-human alkaline phosphatase-conjugated secondary antibody  diluted 1:500, then adding PnPP substrate . Plate readings were done at 405 nm.In order to separate densities with low gradient intensities, representing structurally labile regions, from more structurally rigid regions, we performed edge detection using gradient magnitude calculation and scatterplot region growing to generate a 2D histogram using BioImageXD . Regions were selected to differentiate from low and high gradient magnitudes.C.G.M. and R.H.C. collected/analyzed data and wrote manuscript, L.X. collected/analyzed data, J.H.1, J.H.2, M.B.K. and L.P. analyzed data, O.M.Y. performed assays, Y.S. contributed reagents, L.M., A.V., C.Z., S.W.B. and I.K.S. provided fruitful discussion and analysis, and contributed reagents.