PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for  Beyond its natural functions, RNA has been engineered for diverse applications including therapeutics development, medical diagnostics, and protein engineering.in vitro have a long history. One prominent solution is the pretreatment of samples and solutions with diethylpyrocarbonate (DEPC), which is effective for ribonuclease inhibition.Techniques to mitigate RNA degradation 16. The repeating sulfate units resemble the repeating phosphate units that form the backbone of RNA and are thought to form competitive coulombic interactions with RNase A, thereby occupying its RNA-binding sites and effectively inhibiting RNase A.One promising solution to some of these challenges is the use of inexpensive chemical (non-biologic) RNase inhibitors. Utilizing anionic polymers as a tool for RNase A inhibition is one chemical method that was initially tested over 50 years ago.in vitro applications, such as in vitro transcription (IVT) and coupled and decoupled in vitro transcription and translation. We further analyze the economic viability of using this polymeric RNase inhibitor. Our results suggest that certain applications, particularly RNA storage and in vitro transcription, can benefit from low-cost RNase inhibition through the use of PVSA.Here we describe experiments performed to assess the viability of PVSA beyond RNase A, as an inexpensive, safe, and effective inhibitor against bacterial RNases. We examine PVSA's effects in RNA stabilization in common E. coli lysate in the presence of PVSA. The assays were performed using Ambion's RNaseAlert® assay kit . Inhibition of RNase A (0.75 nM) was examined with increasing concentrations of PVSA (0.001 mg/mL – 50 mg/mL). Consistent with a previous report,50 of 0.15 mg/mL PVSA with greater than 95% inhibition occurring at concentrations greater than 13 mg/mL of PVSA). We also tested the inhibition potency of PVSA against a bacterial lysate from E. coli, which contains diverse bacterial ribonucleases. Many of these intrinsic ribonucleases are known to escape inhibition from biologic RNase A inhibitors.50 of 0.43 mg/mL PVSA  reaction where transcription and translation were coupled. We introduced PVSA at varying concentrations to the coupled in vitro reaction and measured the total green fluorescent protein (GFP) synthesis by its fluorescence  and essentially no protein synthesis was observed at 10 mg/mL PVSA.Next, PVSA's inhibitory capacities were explored in an rescence . As incrin vitro transcription (IVT) using the same plasmid (pY71-sfGFP) and RNA polymerase (T7 RNA polymerase) used in the coupled results above. An aliquot of these reactions was purified by precipitation with isopropanol, and the resuspended mRNA was assessed for storage stability and retained function. Gel electrophoresis suggests IVT reaction products stored for 7 d with 5 mg/mL PVSA had approximately 2 to 4 times the amount of mRNA as those without PVSA.To determine the basis of PVSA inhibition in the CFPS system, the processes of mRNA transcription and translation were decoupled . mRNA enE. coli-based cell-free translation system. The yield of GFP translated from the mRNA transcripts were normalized to the yield of GFP from mRNA produced without PVSA  or the Recombinant RNase inhibitor (Takara). For reference, using 10 mg/mL PVSA in place of the manufacturer's recommended concentration of Murine RNase inhibitor® or Recombinant RNase inhibitor reduces the cost of IVT reagents by more than 95%. Further, we compared the costs per mass of protein produced in a decoupled protein transcription and translation system with adding 10 mg/mL PVSA and without adding PVSA during transcription . Using P20. Thus multiple and diverse RNase inhibitors may be required for protection from multiple sources of RNases. Non-biologic inhibitors offer alternatives that have broader potential due to the lowered specificity of inhibitory interactions and reduced costs  could be performed after removal of PVSA, but is contraindicated in those systems that rely on translation or other RNA-binding proteins.The idea that PVSA can be an inhibitor of RNase A has previously been demonstrated, however, its applications for A comparison of relative yields of protein at different time points  suggests50 values (concentration of inhibitor required to inhibit the reaction by 50%).Ambion's RNaseAlert® kit  comprising a double-labeled oligoribonucleotide with both fluorescent and quenching moieties was used as a substrate for treatment with RNases in the presence or absence of Polyvinyl Sulfonic Acid  as a ribonuclease inhibitor. Cleavage of this RNA derivative substrate molecule separates the fluorophore from the quenching moiety and enables detection of fluorescence, as monitored with a Biotek® Synergy MX plate reader spectrophotometer in fluorimetry mode using Corning® 96 well black plates. Excitation and emission peaks were measured at 480/520 nm respectively. Relative activity for both RNase A and RNase activity in bacterial lysate were measured at varying concentrations of PVSA. The data were normalized and fit to a reciprocal semi-log response curve to determine ICEscherichia coli B strain BL21 Star™ (DE3)  Star™ is rne131 F−BompT gal dcm lon hsdS(Br−Bm−) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12 (λS)). The bacterial cells were grown, harvested, and lysed using an Avestin EmulsiFlex® B-15 Homogenizer with 3 passes at 21,000 psi. The lysate was clarified by centrifugation at 16,000 g, 4°C for 30 min and the supernatant was collected, aliquotted, flash frozen, and stored at −80°C until use. The clarified lysate was then added to the RNaseAlert® reaction at a 0.1% (v/v) concentration. Ribonuclease A  was added to 0.75 nM.Bacterial lysate was prepared as described previouslyIn vitro Transcription (IVT) reactions were prepared with varying concentrations of PVSA. Reactions were prepared with nucleotide triphosphate mixture containing 2 mM each ATP, GTP, CTP, and UTP , T7 RNA polymerase buffer containing 50 mM Tris-Cl (Sigma Aldrich RES3098T-B7) at a pH of 7.5, 15 mM MgCl2 , 5 mM dithiothreitol , and 2 mM spermidine (Sigma Aldrich S2626). A mixture of 3 mM spermidine and 2 mM putrecine (Sigma Aldrich P5780) was also used in the reaction. T7 RNA polymerase enzyme was prepared as described previouslyThe 550 µL IVT reactions were templated using the 50 µg/mL pY71-sfGFP plasmid in vitro transcription/translation reagent costs published previously.Analysis of reagent costs were determined with the prices listed by Sigma Aldrich, Bachem, NEB, and Takara and calculated using other E. coli-based cell-free translation PANOxSP system . The cell-free reactions were then performed as described previously with the exception that plasmid DNA was not added to the reaction.The mRNA transcripts, produced by IVT and purified by precipitation as described above, were then added in equal volume (15 µL) aliquots to an E. coli-based cell-free protein synthesis reactions based on the PANOxSP were performed as described previouslyin vitro translation system described above. As an exception the pY71-sfGFP plasmid, which is transcribed by T7 RNA polymerase in concert with translation, was added instead of adding mRNA transcribed by T7 RNA polymerase from pY71-sfGFP in a prior IVT reaction. The coupled CFPS reaction without PVSA was compared with the coupled system where PVSA was added at varying concentrations (0.0001–100 mg/ml) and the yield was normalized to GFP production without PVSA. Protein production yields of GFP were determined with fluorescence as described above.Here we demonstrate the potential of PVSA as a low cost chemical alternative to biologic RNase inhibitors and present data suggesting its ability to inhibit RNases beyond RNase A. While PVSA is 1,700-fold less expensive and was shown to successfully preserve mRNA, PVSA appears to inhibit mechanisms that involve RNA binding such as translation. To overcome this limitation, the successful removal of PVSA using a simple alcohol precipitation mechanism is reported. It is important to note that this is a proof-of-concept study demonstrating the potential of the chemical RNase inhibitor PVSA, and more head-to-head studies comparing PVSA directly with biologic RNase inhibitors is needed before widespread use. However, here we further report the potential of chemical inhibitors such as PVSA to drastically decrease the costs and complexity of preserving RNA in an RNase ubiquitous world.


Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers. Intestinal epithelial physical barrier plays an important role in protecting organisms from harmful substances in lumen . This moAutophagy is a highly conserved degradation process that removes damaged or aged proteins and organelles to maintain homeostasis in eukaryotic cells. During stress conditions , autophagy is initiated, and subsequently an isolation membrane involved cytoplasmic constituents comes to being a double-membrane structure (autophagosome), which then fuses with lysosomes to form an autophagolysosome for the degradation of the inner membrane and cargo . It has To investigate claudin-2 levels in inflammatory conditions, differentiated Caco-2 cell monolayers were treated with TNF-α (10 ng/mL) for 48 h. Using western blotting analysis, we found that claudin-2 protein levels increased A. In addConsidering that the trend of claudin-2 levels treated by TNF-α was the same as 3-MA, then we explored the status of autophagy in TNF-α induced claudin-2 increase. To evaluate autophagy, we detected the level of LC3B-II and P62 proteins, and the western blotting analysis showed that both the expression of LC3B-II B and P62To figure out the reason of LC3B-II increase induced by TNF-α, autophagy inhibitor 3-MA (5 mM) and autophagic degradation inhibitor Bafilomycin A1 (100 nM) was used before TNF-α treatment. As reported previously, 3-MA decreased the levels of LC3B-II A while bTo further clarify the mechanism, Caco-2 cells were transfected with Ad-mCherry-GFP-LC3B adenovirus. Owing to the fact that green fluorescence was quenched in the acid environment of lysosome, we can only observe yellow and red dots. Yellow dots (merged by mCherry and GFP fluorescence) indicate the autophagosomes that are not fused with lysosome, while red dots (mCherry fluorescense) indicate the compartments that have been fused with lysosome. Thus, the increase of both red and yellow dots indicates the activation of autophagy, while the increase of yellow dots alone means that the degradation process of autophagy is inhibited. As reported previously, bafilomycin A1 treatment, which inhibited autophagosome-lysosome fusion, only markedly increased the number of yellow dots. Similarly, TNF-α treatment largely enhanced the yellow dots while it did not increase the number of red dots C,D, implIt has been shown that an acidic environment is important for the activation of lysosomal proteases in autophagic degradation process . To studTo verify whether PP242 also has impact on the impairment of autophagy flux induced by TNF-α, Caco-2 cell monolayers were administrated with PP242 after TNF-α treatment. Using western blotting assay, we found that PP242 administration relieved P62 increase induced by TNF-α A, but enTo investigate the effect of PP242 on increased claudin-2 expression caused by TNF-α, we used western blotting analysis and immunofluorescence staining to detect claudin-2 expression. Western blotting analysis showed that PP242 alleviated claudin-2 increase caused by TNF-α A. ConsisTo further examine whether the results above extended to other intestinal cell, we repeated experiments above in IEC-6 cell monolayers. First, our experiments showed that TNF-α was significantly enhanced the expression of claudin-2, LC3B-II, and P62 . Then weInflammatory conditions, which are accompanied by intestinal tight junction function changes, are common in IBD and NEC ,6. Pro-iAutophagy is a dynamically catabolic process to maintain cellular homeostasis and provides energy. Studies have shown that autophagy changes when TNF-α administration, but conclusions are contradictory ,23,24,25In the process of autophagic degradation, the acidic condition of lysosome is vital for activation of lysosomal enzymes . Wang etClaudin-2, which belongs to the claudin family of proteins, has been shown to be regulated by a variety of pathways. In this research, we found claudin-2 increases after TNF-α administration which is consistent with those reported by Mankertz et al., who thought phosphoinositide 3-kinase (PI3K) pathway plays an important part in this process . FurtherPrevious study has demonstrated that claudin-2 enhances the intestinal permeability and decreases TER values ,30. SimiTER is a parameter representing the ionic conductance of tight junctions, which mainly reflects the tight junction function . PreviouPP242, a dual mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) inhibitor, has been demonstrated to suppress proliferation and induce apoptosis in tumor cells ,38. PrevIn conclusion, our study reveals that the accumulation of claudin-2 and epithelial tight junction function impairment is partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers. This study provides new insight for the mechanism of claudin-2 increase in TNF-α treated cell monolayers and emphasizes the importance of autophagy in this process, which may promote our understanding of the etiology of inflammatory diseases. In addition, autophagy-lysosome-mediated degradation may be an important pathway to regulate tight junction function which is largely influenced by TJs.2/95% air atmosphere at 37 °C. Recombinant TNF-α was obtained from R&D Systems . Bafilomycin A1  and 3-methyladenine  were purchased from EMD Millipore . PP242 was purchased from Santa Cruz Biotechnology . Claudin-2 antibody , LC3B antibody , P62 antibody , and β-actin antibody  were purchased from relevant manufacturers. Ad-mCherry-GFP-LC3B was purchased from Beyotime Biotechnology .Caco-2 cells and IEC-6 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  and Shanghai Fuxiang Biotechnology  respectively. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were maintained in a 5% CO+ system . The density of specific protein bands was quantified by Image J software .To isolate cellular samples, cells were rinsed with cold PBS twice and lysed in RIPA buffer  containing PMSF. Protein concentration was evaluated by the bicinchoninic acid method using a BCA Protein Assay Kit . Equal amounts of total protein were loaded into SDS-polyacrylamide gels for electrophoresis and transferred to a PVDF membrane . The membrane was blocked in 5% bovine serum albumin (BSA) in PBST for 2 h at room temperature before being incubated overnight at 4 °C with those primary antibodies: β-actin, Claudin-2, LC3B, and P62 antibody. After being reacted with secondary antibody, the membrane was detected with a ChemiDoc XRSCells were grown on 24-well plates and reached 20%–30% confluence at the time of transfection. After two washes, cells were transfected with Ad-mCherry-GFP-LC3B adenovirus at a MOI of 80 in 200 μL DMEM containing 10% FBS for 24 h at 37 °C. Following indicated treatment, autophagy was observed under laser scanning confocal microscope . Autophagy flux was evaluated by calculating the number of yellow and red puncta.Lysotracker Red DND-99  was used for qualitative analysis of Lysosomal pH. Previous study has showed that the red dye becomes more fluorescent in acidic environment while less in alkaline conditions . After dTo quantify lysosomal pH, the method was detailed as previously described ,42,43. ICell monolayers grown on 24-well Transwell chambers  were washed twice with cold PBS and fixed with 4% Polyoxymethylene for 15 min at room temperature. Then the cell monolayer was incubated with 3% BSA for 30 min at room temperature and labeled with claudin-2 antibody overnight at 4 °C. After being washed with PBS, cell monolayers were incubated with Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG for 1 h at 37 °C. After two more washes, the cell monolayer was incubated with DAPI nuclear stain solution for 10 min. Fluorescence images were captured by inverted fluorescence microscopy (Leica).5 cells/mL for up to 14 or 6–7 days to form a cell monolayer, respectively. To evaluate the TER of each cell monolayer, the mean resistance value of unseeded Transwell inserts was subtracted from each value, and corrected for the area of the filter (0.33 cm2).All experiments were carried out as previously reported ,44,45. IPermeability studies were performed with FITC-Dextran . Cells were grown on the filters in a 24-well Transwell chamber. After drug treatment, the media in the basolateral and apical compartment of the Transwell chamber was replaced with 0.6 mL DMEM or 0.1 mL DMEM containing FITC-Dextran at 1 mg/mL. After 1 h incubation, medium from the basolateral compartment was used for spectrofluorometric measurement of Dextran concentration.t-test (two-tailed) for two group comparison or one-way ANOVA with Tukey’s Multiple Comparison test for more than two groups by GraphPad Prism 6.0 software. p < 0.05 was set as statistically significant.All data are shown as mean ± SD of at least triplicate experiments. Statistical difference was estimated using Student’s unpaired Our study reveals that TNF-α-induced claudin-2 increase and impairment of epithelial tight junction function are partly attributed to the inhibition of autophagic degradation in vitro. Further research is needed to evaluate other tight junctions that may play a role in this process. This research may provide a new insight for our understanding on the etiology of some inflammatory diseases.


HIV vaccine able to elicit broadly neutralizing antibodies (bNAbs) remains a major challenge. Antibodies that recognize the V2 region at the apex of the HIV envelope trimer are among the most common bNAb specificities during chronic infection and many exhibit remarkable breadth and potency. Understanding the developmental pathway of these antibodies has provided insights into their precursors, and the viral strains that engage them, as well as defined how such antibodies mature to acquire breadth. V2‐apex bNAbs are derived from rare precursors with long anionic CDR H3s that are often deleted in the B cell repertoire. However, longitudinal studies suggest that once engaged, these precursors contain many of the structural elements required for neutralization, and can rapidly acquire breadth through moderate levels of somatic hypermutation in response to emerging viral variants. These commonalities in the precursors and mechanism of neutralization have enabled the identification of viral strains that show enhanced reactivity for V2 precursors from multiple donors, and may form the basis of germline targeting approaches. In parallel, new structural insights into the HIV trimer, the target of these quaternary antibodies, has created invaluable new opportunities for ontogeny‐based immunogens designed to select for rare V2‐bNAb precursors, and drive them toward breadth.The development of a preventative  Immunological Reviews.This article is part of a series of reviews covering B cells and Immunity to HIV appearing in Volume 275 of 1The design of an effective preventative HIV vaccine continues to represent a major public health challenge, with 36 million people currently living with HIV. Despite more than 17 million people now accessing antiretroviral treatment globally and an array of prevention tools, new infections continue to occur at high rates, particularly in parts of southern Africa, with 2 million new infections estimated to occur annually. Many successful vaccines rely on the elicitation of neutralizing antibodies to block viral entry and provide sterilizing protection. However, this is a particular challenge for HIV, which is among the most variable and heavily glycosylated viruses known. Several vaccines have been tested with little or no efficacy, and none of these vaccines have been able to elicit the types of broadly neutralizing antibodies (bNAbs) that will be required to be effective against the enormous global diversity of HIV. Despite these setbacks, there is strong rationale for pursuing bNAbs to prevent HIV infection. Passive immunization of bNAbs isolated from infected donors has long been known to protect non‐human primates from infection [reviewed in 2The HIV‐1 envelope (Env) glycoprotein complex, which consists of a heterotrimer of three molecules of gp120 and three molecules of gp41, is responsible for mediating viral entry into host cells, and is the sole target of neutralizing antibodies. The first and second variable regions (V1V2) of gp120 are located at the apex of the envelope trimer, and are highly variable in terms of sequence, glycosylation and length, largely due to mutations and insertions in two regions, in the middle of V1 and toward the C‐terminal end of V2  glycans at N156 and N160. ZM109 falls among a minority of HIV‐1 strains that do not have a glycan at N156 but instead contains a glycan at residue N173, which is in close three‐dimensional proximity to N156 in the structure and serves as a viable substitute. The co‐crystal structure of PG16 with a scaffolded V1V2 domain from ZM109, grown in cells treated with the α‐mannosidase II inhibitor swainsonine (predicted to induce hybrid‐type glycans), highlighted a difference in glycan specificity for this clonal relative of PG9. These preferences suggest that while glycans at residues N156/N173 and N160 may primarily consist of Man5,PG9‐bound V1V2 revealed how bNAbs could target this region not only by penetrating the glycan shield, but by using the dense array of glycans, typically regarded as a viral defense, as part of their epitope. PG9 neutralization is dependent upon glycans at residues N156 (or N173 in some strains) and N160, with the latter conserved glycan being critical for recognition  and residue 181. This was supported by the isolation of two V2‐directed antibodies, called CH58 and CH59, both isolated from RV144 vaccines. These antibodies, despite apparently targeting conserved elements of V2, did not display broadly neutralizing activity.4Broadly neutralizing antibodies that target the V2 region prefer or require a quaternary epitope,Insights into the quaternary requirements for V2‐directed bNAbs and difficulty with crystallizing an asymmetric complex (containing 1 Fab per trimer) led to the screening of trimeric scaffolded V1V2 proteins as tools for crystallization to understand the atomic‐level details of lineages other than PG9/PG16. Through ELISA screening on V1V2 trimeric scaffolds in a 96 well format, a scaffold that bound to V2 bNAbs better than monomeric gp120s was identified. A 3.1 Å structure of CH03 (from donor CH0219) on a trimeric V1V2 scaffold, derived from the virus that superinfected donor CAP256 (described in more detail below), showed a very similar mechanism of engagement to that of PG9Since V2‐directed antibodies prefer or require a quaternary epitope, advances in the construction of a soluble trimeric molecule that recapitulates the architecture of the native trimeric viral spike were critical to understanding the nature of the epitope. A crystal structure and high resolution EM structure of BG505 SOSIP.6645Studies of the ontogeny of other classes of bNAbs have proven to be extremely revealing. In particular the shared germline gene structures and predictable developmental pathways for the CD4 binding site (CD4bs) class of bNAbs have enabled great progress in the development of germline targeting immunogens for this class (reviewed elsewhere in this series). Similar studies have recently been performed for V2‐apex antibodies, which based on commonalities in structure  have been proposed to form an “extended class” of antibodiesSuch studies rely on the comparison of V2‐apex antibodies from multiple donors. The majority of V2 bNAbs were isolated from donors during chronic infection, but despite the absence of early sequences from these antibody lineages, have proved highly informative. For the PG9/PG16 mAbs, little additional lineage information is available and approximations of early members can only be calculated through comparison of the two antibodies and their germline VH/VL genes. In these constructs, the VH or the VH and VJ gene segments are replaced by the corresponding germline genes, however, the CDR H3, a major determinant of neutralization, which spans the V‐D‐J junction, cannot be accurately reverted and is left as the mature form. These germline reverted constructs  approximate the properties of a true UCA as well as possible. One version of the reverted PG9 gHgL bound to eight trimeric envelope strains in an ELISA format and neutralized four of 182 strains.Similarly for CH01‐CH04, two versions of a putative precursor (RUA‐1 and RUA‐2) were inferred from the complete sequences of the four lineage members (CH01‐CH04) isolated from donor CH0219 during chronic infection.The same approach was taken with the PGT145/PGDM1400 lineage isolated from donor IAVI84, which differs slightly from the other V2 bNAbs as its CDR H3 is extended at less of an angle and the approach of the antibody to the V2 domain is more centered. Given this, the exact binding mode may differ from the other V2 bNAbs, however, it shares the same genetic characteristics observed in the others. The CDR H3s of most members are 32 residues in length although some, including PGT145, contain a single amino acid deletion. Intradonor phylogenetic analysis of sequences from the mature isolated antibodies as well as NGS data revealed the earliest intermediate that could be well‐defined still had ~6% nucleotide‐level SHM. The CDR H3 had 32 amino acids and a net −5 charge, and did not include sulfated tyrosines, suggesting that many of the determinants of the canonical long, anionic CDR H3 were formed early in the lineage.Of all the V2 bNAb lineages, only the CAP256‐VRC26 family isolated from a subtype C infected and superinfected donor has been characterized longitudinally from the time of infection, providing key insights that support and extend the studies described above. The earliest cloned antibody, CAP256‐VRC26.01, was isolated at week 59 and 32 other members of the lineage were isolated at weeks 119‐206 by a combination of B cell culture and trimer‐based sorting.610 and 1011 B cells in vivo, it is likely that as for other rare bNAb precursors,The long CDR H3s that characterize V2‐apex antibodies range from 24‐37 amino acids (Kabat numbering). These are in contrast with typical antibodies, which have a mean length of 14‐16 amino acids.Studying the neutralization and envelope binding profiles of V2‐apex antibody precursors may provide clues as to the nature of the bNAb‐initiating virus that triggered these lineages. Overall, studies of CAP256 and of other lineages suggest that V2‐apex UCAs are unusual clones selected from the naïve repertoire, which do not neutralize or bind the vast majority of strains. Indeed, in CAP256 for which the most accurate V2‐apex precursor has been determined, the UCA neutralized only the superinfecting transmitted/founder virus, but not the primary transmitted/founder virus or any other virus from a panel of two hundred.Identifying additional such V2‐apex bNAb‐initiating envelopes has potential utility for immunogen design, but relies on detailed longitudinal sampling, and on these envelopes being generally more reactive with multi‐donor V2‐apex precursors than other viral envelopes. Previous studies of V2‐apex antibodies had sought to identify germline‐reactive envelopes for individual lineages as the basis of future immunogens.7Beyond the initial engagement of precursors with long CDR H3, a key recent focus has been on how V2‐apex antibodies (and other bNAb specificities) mature to acquire breadth. Longitudinal analyses of both antibody and virus during maturation of the CAP256‐VRC26 lineage have highlighted the substantial role of viral diversification in the emergence of neutralization breadth.Comparison of the neutralization capacity of mAbs isolated from both branches against longitudinally sampled autologous viruses provided a mechanism for these distinct evolutionary outcomes. The antibodies in the “dead‐end” branch were unable to tolerate viral escape mutations that were selected as the viral quasispecies evolved to evade the early bNAb responsesThese studies of bNAb maturation in donor CAP256 have defined a mechanism for how accumulating viral variants contributed to V2‐apex bNAb development. Viral escape from early members of the antibody lineage creates multiple viral immunotypes. In parallel, SHM generates antibodies with differential abilities to engage these epitope variants. Those antibody sublineages that are able to tolerate variability at key epitope contacts may develop breadth. These findings have implications for ontogeny‐based immunogen design, suggesting that breadth may require the incorporation of multiple immunotypes into vaccines, to drive antibody maturation toward tolerance of diversity8Studies of the ontogeny of V2‐apex antibodies have led to increased efforts to incorporate structural and virological findings into targeted immunogens. Many clinical trials based on immunization with monomeric gp120 have failed to elicit bNAbs, including those directed to the V2‐apex.The advance of soluble trimers such as BG505 SOSIP.664 has overcome many of the obstacles associated with monomeric immunogens and therefore marks a critical and exciting advance toward targeting V2‐directed bNAbs. The quaternary nature of V2‐directed bNAbs elicited through natural infection strongly suggest that effective immunogens targeting this ontogeny need to present the full epitope present on the native viral spike. Availability of this soluble trimer has provided a great deal of structural information (described above) recently enabling the optimization of immunogen design targeting specific epitopes, particularly those of a quaternary nature. Atomic‐level details from multiple structural studies of this construct has facilitated additional stabilizing mutations such as a disulfide between residues 201 and 433 that prevents the trimer from conformationally shifting upon binding of CD4. However, providing a stabilized architecture of the trimeric V1V2 epitope may not be sufficient to elicit a response. Effective immunogens must also take into account the distance and orientations of each V1V2 domain relative to each other, the equilibrium of the domain at the cap (the V1V2 domain may shift as observed by smFRETThe identification of a few strains that have a higher probability of engaging V2‐apex bNAb precursors from multiple donors, described above,9With the availability of a soluble trimer that mimics that native spike, a great many ideas for immunogen design that were technologically unavailable just a few years ago are now possible. An explosion of crystal and EM structures


From the frightening “publish or perish” to the stimulating “publish and flourish”, theadvice remains the same: an academic career depends on writing up the results ofresearch and making them public. Where to publish also hinges on the idea ofbroadcasting the findings, and it is generally agreed that manuscripts should be firstsubmitted to the most renowned periodicals. There are many reasons for this consensus,among them the likelihood of the manuscript being revised by important researchers inthe field and, if accepted, the receipt of a kind of seal of quality. Last but notleast, to be published by an important periodical increases the chances of being citedbecause these are read by the most productive researchers. Subsequent citation in otherpapers is the end goal and a measure of the quality of the contribution. This is theuniversal path in the search for excellence in academic life. Often times an outstandingpublication paves the way for more in-depth cooperative research and has a decisiveimpact on academic careers.Dementia &Neuropsychologia was to bring these papers to light and to make suchoccurrences a thing of the past. Since the advent of electronic online publication ofscientific papers, it has become easier to make results public and to retrieve paperspublished almost anywhere in the world.However, manuscripts frequently present findings that are important only to a specificregion or to speakers of a particular language. Moreover, papers may be more relevant tocertain cultural groups than others thereby reducing their chances of publication in aprominent periodical. In the past, most of these manuscripts ended up being published inobscure local journals, often not in English. In this sense, they were never really madepublic. One of the worst upshots of the ostracism of these papers was the realizationthat sometimes the same type of research was later conducted by other investigators,who, unaware of the previous papers, repeated the same methodological errors criticizedby these earlier papers. One of the main reasons for launching Dementia & Neuropsychologia is the official journalreceive a printed copy of each issue, but what about the scientific community elsewhere?Dementia & Neuropsychologia is accessible online at www.demneuropsy.com.br, where papers are freely available, and the dataon those accessing the site and downloading papers may provide a clue to the visibilityof the journal.It is now fitting to inform our authors and readers on the exposure of papers publishedby our journal. Are they really reaching the scientific community? Members of thesocieties for which Since December 2008, when only 136 downloads of manuscripts were registered, there hasbeen a steady rise in number of visits with downloads reaching 2,315 in October 2009,giving a grand total of 11,022 in the past 11 months. The visitors were from 66countries, with the most frequent countries of origin of the visitors being, indescending order of number of visits: Brazil, the United States, China, Portugal, theUnited Kingdom, Australia, the Netherlands, Italy, Germany, Belgium, Japan, the RussianFederation, Argentina and India.Dementia &Neuropsychologia.These numbers are truly rewarding for our authors and may serve as an incentive forformer and future authors to submit their papers to Ricardo NitriniEditor-in-Chief


Importantly, the three-dimensional orientation of the scTNF trimers impacts the bioactivity of the oligomerized scTNF ligands. Our data unravel the organization of tmTNF-mimetic scTNF ligands capable of robustly activating TNFR2 and introduce novel TNFR2 agonists that hold promise as therapeutics to treat a variety of diseases.Tumor necrosis factor receptor 2 (TNFR2) is known to mediate immune suppression and tissue regeneration. Interestingly, the transmembrane form of tumor necrosis factor (tmTNF) is necessary to robustly activate TNFR2. To characterize the stoichiometry and composition of tmTNF during TNFR2 activation, we constructed differently oligomerized single chain TNF ligands (scTNF) comprised of three TNF homology domain (THD) protomers that mimic tmTNF. Using a variety of cellular and  This structural composition leads to the self-association of TNF monomers into trimers and is necessary for receptor binding3. Due to the carboxy-terminal localization of the THD, both the transmembrane form as well as soluble TNF ligands assemble into trimers. However, the THD-mediated receptor interaction alone is not necessarily sufficient to activate receptor-associated intracellular signaling pathways. For several members of the TNF receptor superfamily, the initial formation of ligand receptor complexes is followed by secondary multimerization into supramolecular clusters7.The tumor necrosis factor (TNF) superfamily is a family of different cytokines with various functions. Most ligands of the TNF family are synthesized as trimeric type II transmembrane proteins that can be released into a soluble form via proteolytic processing. The structural hallmark defining the TNF ligand family is the carboxy-terminal TNF homology domain (THD) which is composed of two stacked β-pleated sheets that adopt a conserved jellyroll-like tertiary fold8. Trimeric sTNF tends to irreversibly dissociate at subnanomolar concentrations, thereby losing its bioactivity9. Dissociation and thus inactivation can be prevented by connecting three TNF monomers with short intramolecular peptide linkers, resulting in covalently stabilized single-chain TNF trimers (scTNF)10.Despite their similar trimeric organization, membrane-bound and soluble TNF ligands can differ in their activity. This difference is specifically obvious for the name-giving family member TNF. TNF is synthesized as a trimeric transmembrane protein  that can be released into soluble circulating TNF homotrimers  via cleavage of the ectodomain by TNFα-converting enzyme (TACE/ADAM17)13.TNF can bind two structurally distinct transmembrane receptors, TNF receptor (TNFR) 1 and TNFR2, which have marked differences in expression patterns, structure, signaling mechanisms and functions14. Different association/dissociation kinetics of the ligand/receptor complexes may contribute to the different TNFR activation capabilities of sTNF and tmTNF. Whereas sTNF has a remarkably high affinity for TNFR1 (Kd = 1.9 × 10−11 M), the affinity for TNFR2 is significantly lower (Kd = 4.2 × 10−10 M)15. The high affinity of sTNF for TNFR1 is mainly caused by stabilization of ligand/receptor complexes, while transient binding of sTNF to TNFR2 results in short-lived signal incompetent complexes16.Both tmTNF and sTNF can activate TNFR1 in the picomolar range, whereas TNFR2 is only robustly activated by tmTNF17. In this line, oligomerization of soluble ligand trimers, e.g. via antibody-mediated crosslinking, does not increase activation of TNFR118, whereas secondary oligomerization of soluble TNF trimers converts this molecule into an active TNFR2 agonist20.A probable reason for the tmTNF-dependence for TNFR2 activation is the higher demand of TNFR2 for ligand-mediated crosslinking to allow signaling cluster formationTo characterize the stoichiometry and composition of tmTNF necessary to efficiently activate TNFR2, we genetically engineered differently oligomerized TNFR2-selective scTNF ligands. The activity of these fusion proteins was then compared in regard to TNFR2 binding, TNF/TNFR2 complex formation and induction of specific cellular responses.R2 mimics tmTNF and efficiently activates TNFR221. We, therefore, fused a mouse TNFR2-specific (D221N/A223R) sc-mTNF (sc-mTNFR2) to different oligomerization domains, resulting in fusion proteins with different arrangements of the sc-mTNFR2 moieties , the tetramerization domain of p53 (aa 320–359)25 that exhibits an antiparallel arrangement of the domains (dimer of dimers) resulting in a tetrahedron-like 3D structure, and GCN4 (aa 249–281), a mutated helix from the yeast transcription factor GCN427, with a parallel arrangement of the four coiled-coil domains28 , 70 kDa (EHD2-sc-mTNFR2) and around 55 kDa (p53-sc-mTNFR2 and GCN4-sc-mTNFR2), matching the calculated molecular mass of 50.2 kDa, 66.6 kDa, 59.7 kDa and 58.9 kDa for sc-mTNFR2, EHD2-sc-mTNFR2, p53-sc-mTNFR2 and GCN4-sc-mTNFR2, respectively. Under non-reducing conditions, an additional band of approximately 140 kDa (dimer) was observed for EHD2-sc-mTNFR2, indicating the disulfide-stabilized dimer formation via the EHD2 domain. Both p53-sc-mTNFR2 and GCN4-sc-mTNFR2 showed only partially oligomerized bands, which was expected since these oligomerization domains are not stabilized via covalent disulfide bonds. The oligomerization state of the fusion proteins was further confirmed by SEC , dimers  or tetramers .Previously, we demonstrated that oligomerized, covalently stabilized scTNFies Fig. , Fig. S1s28 Fig. . All fuss28 Fig. . Under rSEC Fig. . All fusR2 muteins bound to TNFR2. Whereas the monomeric sc-mTNFR2  only interacted with TNFR2 at high concentrations, similar to recombinant soluble TNF (sTNF), EC50 values of EHD2-sc-mTNFR2 , p53-sc-mTNFR2  and GCN4-sc-mTNFR2  were significantly lower, with GCN4-sc-mTNFR2 displaying the highest binding efficiency  was determined for sc-mTNFR2. Minor differences were observed between EHD2-sc-mTNFR2, p53-sc-mTNFR2 and GCN4-sc-mTNFR2, with GCN4-sc-mTNFR2 showing the lowest off-rate. We, therefore, investigated, if differences between EHD2-sc-mTNFR2, p53-sc-mTNFR2 and GCN4-sc-mTNFR2 can be detected using a low density chip (130 Hz), with non-saturated immobilized TNFR2  with saturated immobilized TNFR2, strong binding of sc-mTNFR2 and GCN4-sc-mTNFR2 had lost affinity for TNFR1 and the capacity to activate this receptor.TNFR2 selectivity of the oligomerized TNF muteins was confirmed using HeLa and L929 cells. In contrast to wild-type TNF used as a positive control, none of the TNFR2-selective fusion proteins induced TNFR1-dependent IL-6 secretion in HeLa cells Fig. . Further29 and efficient signal initiation requires the formation of larger ligand/receptor complexes30. We, therefore, investigated the differences between the oligomeric TNF muteins to induce TNF/TNFR2 signaling cluster formation. Mouse BV-2 microglia cells were incubated with different concentrations of the fusion proteins and TNF/TNFR2 cluster formation was visualized via immune fluorescence  and TNFR2 (red) signals. Around 50% less TNF/TNFR2 signaling complexes were induced with p53-sc-mTNFR2. Using a high concentration of 1 nM, similar efficient TNF/TNFR2 cluster formation (90–100%) was observed after incubation with p53-sc-mTNFR2 and GCN4-sc-mTNFR2. In contrast, both scTNFR2 and EHD2-sc-mTNFR2 were less capable of inducing TNF/TNFR2 cluster formation, with 30–40% clusters formed at the highest concentration. Notably, at low concentrations (0.1 nM), almost no cluster formation was observed with sc-mTNFR2, whereas EHD2-sc-mTNFR2 induced approximately 20% cluster formation.Receptors of the TNF family are activated by ligand-mediated oligomerizationnce Fig. . At low R2-induced secretion of Cxcl-2 in BV-2 cells. Here, GCN4-sc-mTNFR2  was 5-times more potent than p53-sc-mTNFR2   was observed, whereas sc-mTNFR2 induced the weakest Cxcl-2 secretion.We then investigated if the elevated binding capacity of the dodecavalent TNF oligomers is also converted to an increased bioactivity. First, we determined TNF31. Therefore, cells were incubated with the fusion proteins for 24 hours and cell viability was measured using the crystal violet assay. Again, bioactivity was increased with higher oligomerization of the TNF muteins  was just slightly more potent than EHD2-sc-mTNFR2 , GCN4-sc-mTNFR2 induced a more than 3-times increased response  compared to EHD2-sc-mTNFR2.Next, we investigated TNFR2-induced proliferation of mouse thymocytes. Therefore, cells were incubated in presence of the different fusion proteins for 4 days and proliferation was determined by measuring their metabolic activity. All oligomerized fusion proteins induced proliferation. Whereas sc-mTNFion Fig. . Whereas34. We choose to use a concentration of 0.3 nM of the fusion proteins, due to the different effects observed in the previous assays for this concentration. Similar results were obtained for both, TNFR2-induced expansion of human CD25+HLA-DR+ 34 and TNF is known to expand and stabilize Tregs via TNFR237, we then investigated the potential of the different sc-mTNFR2 fusion proteins to expand Tregs. Therefore, CD3+ mouse T cells were activated using αCD3, cultivated in presence of the tmTNF mimetics and the number of CD4+CD25+FoxP3+ Tregs was quantified by flow cytometry , known to express TNFR2DR+ Fig.  and mousR2+ Fig.  activatetry Fig. . Similarin vivo application of a TNFR2 agonist leads to the expansion of Tregs and protects mice from acute graft versus host disease38. Therefore, we administered low concentrations (1 mg/kg) of the TNF muteins EHD2-sc-mTNFR2, p53-sc-mTNFR2 and GCN4-sc-mTNFR2 in mice and determined the number of CD4+CD25+FoxP3+ Tregs in the spleen after three days  EHD2-sc-mTNFR2 induced an efficient expansion of Tregs, too . Antibody-mediated multimerization of Flag-tag-containing variants of soluble TNF, CD95L, TRAIL, and CD40L with Flag-specific antibodies resulted in a dramatic increase in avidity and efficient activation of their cognate receptors TNFR2, CD95, TRAILR2, and CD40, respectively10 and TNFR247, oligomerization of trimeric TNF ligands is highly superior in activating TNFR221. In line with this, additional data indicate that mere mechanical fixation of TNF to an immobilized surface is insufficient to efficiently activate TNFR2. Rather, a certain density of the ligand and an additional stabilization of TNFR2 by cluster formation seems to be mandatory for efficient activation48. Whereas data exist that oligomerization of a trimeric scTNF ligand improves its bioactivity, no data were available on the possible stoichiometry and composition of oligomerized TNF ligands necessary to robustly activate TNFR2.The dependence of the naturally occurring transmembrane ligand or membrane-mimetic variants for robust activation is especially obvious for TNFR2. Whereas it was shown that trimer stabilization both improves activation of TNFR1R2 and GCN4-sc-mTNFR2 both bind more stable to TNFR2 compared to the hexavalent EHD2-sc-mTNFR2. Furthermore, using different cellular response assays, GCN4-sc-mTNFR2 consistently exerted the highest bioactivity. In addition, in all assays a slightly increased bioactivity of p53-sc-mTNFR2 compared to EHD2-sc-mTNFR2 was observed. A first important step for efficient signal induction is the formation of higher order TNF/TNFR2 signaling clusters. Our microscopical analyses showed that GCN4-sc-mTNFR2 efficiently promoted the cluster formation even at low sub-nanomolar concentrations. Likewise, p53-sc-mTNFR2 was superior in cluster-formation compared to EHD2-sc-mTNFR2, which translated into a 3.5-fold higher bioactivity in a Cxcl-2 chemokine secretion assay with BV-2 cells.Receptor binding studies revealed that the dodecavalent variants p53-sc-mTNFR2 shares the highest similarity with the assumed natural tmTNF configuration, since all TNF trimers in the oligomer are arranged in a way that they point into the same direction. In contrast, p53-sc-mTNFR2 seems to have more a spider-like shape in which the binding sites point into the directions of a tetrahedron24. We, therefore, hypothesize that these different 3D-structures mainly impact the distance between the scTNF units, with the highly potent GCN4-sc-mTNFR2 configuration displaying the least distance between the scTNF units. Therefore, it is likely that the distance between TNF trimers in tmTNF or tmTNF mimetic ligands also plays a role for the bioactivity. Interestingly, the in vivo application of the tetramers p53-sc-mTNFR2 and GCN4-sc-mTNFR2 is highly superior to dimeric EHD2-sc-mTNFR2, whereas no significant differences between p53-sc-mTNFR2 and GCN4-sc-mTNFR2 were observed. This superior bioactivity of p53-sc-mTNFR2in vivo compared to the in vitro assays might be due to possible cis and trans activation of TNFR2 on the same and on neighboring cells, respectively. We propose that a p53-sc-mTNFR2 molecule that binds to TNFR2s with a minimum of two scTNFs is capable of activating TNFR2, similar to EHD2-sc-mTNFR2, but still possesses two free scTNFs capable of activating TNFR2 in trans on other cells. Keeping in mind that TNF/TNFR2 clusters are formed, such a higher order p53-sc-mTNFR2/TNFR2 cluster with free scTNF molecules could mimic natural tmTNF signaling, i.e. an additional transactivation of a juxtaposed cell.Altogether, our data show that the three-dimensional orientation of tmTNF-mimetic TNF ligands is important and determines the efficiency of TNFR2 signal induction. Our data suggest that the configuration of GCN4-sc-mTNF50. Drug potency and efficacy is of particular interest for therapeutic applications, as lower doses of the particular drug are needed to induce maximal therapeutic effects. Therefore the high and specific bioactivity of tmTNF-mimetic tetrameric scTNF oligomers represent a novel approach towards TNFR2-selective drugs.Since TNFR2 plays an important role in immune regulation and tissue regeneration, ligands that effectively activate TNFR2 hold great promise as novel therapeutics to treat a variety of diseases, including inflammatory and neurodegenerative diseasesin vitro and in vivo model systems. Our findings, therefore, are of principal relevance for development of TNFR2 agonists for potential therapeutic application. For the use in humans completely human molecules are needed. This can be readily achieved by exchanging the sc-mTNFR2 part to its human counterpart scTNFR2 which was previously described by our group21. The yeast GCN4 tetramerization domain was used here as a model of a coiled-coil structure. To circumvent potential immunogenicity of foreign protein domains, this domain could be exchanged to similar coiled-coil tetramerization domains of human proteins. In addition, other modules and formats resulting in a GCN4-sc-mTNFR2-like configuration are possible to design highly potent TNFR2 agonists.In conclusion, we show that controlled oligomerization of scTNFs is a prerequisite to efficiently activate TNFR2. By introducing various oligomerization domains to generate dimeric or tetrameric scTNF ligands with different three-dimensional orientation of the scTNF units, we identified formats suitable for efficient TNF/TNFR2 complex formation and signal induction and high bioactivity in In summary, our data show that TNFR2 activation by dodecavalent ligands is clearly superior to trivalent or hexavalent ligands. We also demonstrate that the predicted 3D-structure of a dodecavalent TNFR2-selective TNF mutein appears to be important for the bioactivity of the ligand. In addition, because of conserved structural features of the members of the TNF/TNFR superfamily, our findings suggest that similar conditions exist for other members of this biologically important cytokine family.14. The anti-TNF antibody (clone F6C5) was from GeneTex  and anti-TNF (HP8001) from Hycult Biotech . The anti-CD3 (clone UCHT1 & 17A2) and anti-TNFR2 antibody (AF-426-PB) were from R&D Systems . Fluorescence-labeled antibodies against CD3, CD4, CD25, HLA-DR, TNFR2 and FoxP3 were from Miltenyi Biotech . Secondary antibodies coupled to Alexa Fluor 488 or Alexa Fluor 546 were from Life Technologies  and horseradish peroxidase (HRP)-labeled anti-mouse IgG antibodies were purchased by Jackson ImmunoResearch Laboratories . Recombinant interleukin 2 (IL-2) was purchased by Immunotools . Actinomycin D, 4′,6-Diamidin-2-phenylindol (DAPI) and 3-−2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma-Aldrich and 3,30,5,50-tetramethylbenzidine (TMB) substrate was purchased from Biolegend . All other chemicals were of analytical grade.The TNFR2-specific antibody 80M2 has been described21. Briefly, HEK293-6E cells, grown in F17 medium , were transiently transfected with expression constructs of TNF muteins using polyethyleneimine (Sigma). The day after, Tryptone N1  was added to the cell culture and cells were cultivated for additional 4 days. Then, supernatant was collected and recombinant proteins were purified by immobilized metal ion chromatography (IMAC). For this purpose, supernatant was loaded onto a column containing Ni-NTA agarose  and unbound proteins were washed away using IMAC wash buffer (50 mM sodium-phosphate-buffer). Bound proteins were eluted with IMAC elution buffer  and dialyzed  against PBS overnight at 4°C. Finally, eluted proteins were purified by SEC. Protein concentration was determined by measuring the absorbance at 280 nm. For coomassie staining, 2 µg of the purified TNF muteins were denatured in Laemmli buffer, resolved by 8% SDS-PAGE and stained with coomassie.Production and purification of recombinant proteins was described previouslyApprox. 20 µg protein was applied to a BioSep-SEC-S2000 column  equilibrated with PBS and eluted at a flow rate of 0.5 ml/min. For determining the size of recombinant proteins, standard proteins were run under the same conditions.2SO4 and the absorbance at 450 nm was determined with an absorbance reader  and data were analyzed using the software Microsoft Excel and GraphPad Prism 4 . Between each step, non-bound proteins were removed by washing 4 times with 0.005% Tween-20 in PBS.ELISA plates  were coated with TNFR1-Fc or TNFR2-Fc fusion proteins at 1 µg/ml in PBS and incubated at 4°C overnight. Residual binding sites were blocked with 2% skim milk powder in PBS at RT for 2 hours. TNF muteins were diluted in 2% skim milk powder in PBS and incubated for 1 hour at RT. Bound proteins were detected with mouse monoclonal antibodies to TNF  and HRP-conjugated anti-mouse IgG antibodies , followed by incubation with TMB substrate solution. Reaction was stopped by addition of 1 M HAffinities of the oligomerized fusion proteins for TNFR2-Fc were determined by quartz crystal microbalance measurements . Therefore, TNFR2-Fc fusion proteins were chemically immobilized on a carboxyl sensor chip according to the manufacturer’s protocol at a high (270 Hz) and low (130 Hz) density, respectively. Binding experiments were performed in PBST  pH 7.4 at a flow rate of 25 ml/min at 37 °C. The chip was regenerated with 10 mM Glycine HCl pH 2.0. Before each measurement, a baseline was measured which was subtracted from the binding curve. Data were collected using Attaché Office software  and TraceDrawer .4 cells/well) were grown in 96-well flat bottom cell culture plates overnight. L929 cells were treated with actinomycin D (1 µg/ml) and Kym-1 cells with 80M2 (1 µg/ml) for 30 minutes prior to addition of TNF muteins. L929 and Kym-1 cells were incubated with different concentrations of TNF muteins for 24 hours at 37 °C. Cells were washed with PBS and incubated with crystal violet  for 20 minutes to stain viable cells. The dye was washed away under rinsing water and cells were air-dried. Crystal violet was resolved with methanol and the optical density at 550 nm was determined. Each sample was analyzed in triplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism .L929 or Kym-1 cells  or IL-6  according to the instructions of the manufacturer. For determination of inflammatory marker CRP in the blood, whole blood was withdrawn and analyzed by an ELISA specific for mouse CRP  according to the instructions of the manufacturer. The absorbance at 450 nm was determined and the amount of released Cxcl-2 or IL-6 was determined with the provided standard and calculated using the software GraphPad Prism.g, 5 min) and washed once with culture medium . Then 1.5 × 105 cells were plated into αCD3 coated  96-well (U) plates and cultivated for 4 days in presence of the oligomerized fusion proteins. Number of cells was determined by MTT −2,5-Diphenyltetrazolium Bromide) assay. Therefore, cells were incubated with MTT (0.5 mg/ml) for 2 hours at 37 °C. Then lysis buffer  was added, cells were lyzed overnight and optical density at 550 nm was determined. Each sample was analyzed in triplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism.Thymus of C57BL/6 mice was isolated and mashed through a 40 µm cell strainer (Flacon). Cells were centrifuged .g without brake. Interphase, including mononuclear cells of peripheral blood (PBMCs) was removed and washed with 30 ml RPMI . For removal of platelets, cells were resuspended in 40 ml RPMI and centrifuged for 5 min at 200 g. Then, CD3+ T cells were isolated by magnetic separation using the Pan T Cell Isolation Kit (Miltenyi Biotech). Purified T cells were plated in X-Vivo 15 medium (Lonza) in αCD3 coated (6 h at 4 °C) 96-well (U form) plates for T cell activation. Cells were incubated in presence of IL-2 and the oligomerized fusion proteins for 4 days. Then surface expression of CD25 and HLA-DR was determined by flow cytometry according to manufacturer’s instructions . At least 20,000 cells per sample were acquired. Data were acquired using a MACSQuant Analyzer 10 (Miltenyi) and analyzed with FlowJo .Blood of volunteer human donors was diluted 1:2 with RPMI medium. Then 30 ml diluted blood was layered over 10 ml Histopaque-1077 (Sigma-Aldrich) and centrifuged for 20 min at 800 g, 5 min) and washed once with 10 ml MACS buffer. Then CD3+ T cells were isolated using the FACS Aria III and plated in αCD3 coated (6 h at 4°C) 96-well (U form) plates for T cell activation. T cells were cultivated in presence of IL-2 and the oligomerized fusion proteins for 4 days. Then expression of CD25, TNFR2 and/or FoxP3 was determined by flow cytometry according to manufacturer’s instructions . Data were acquired using a MACSQuant Analyzer 10 (Miltenyi) and analyzed with FlowJo .Spleens from C57BL/6 wildtype mice were dissociated through a 40 µm cell strainer and collected in 10 ml MACS buffer . Splenocytes were centrifuged  and incubated in 3 ml RBC buffer  per spleen for 5 minutes at room temperature to lyse red blood cells. Then 10 ml MACS buffer was added and splenocytes were centrifuged for 5 min at 300 g. Cells were washed once with 10 ml MACS buffer  and afterwards collected in MACS buffer. Expression of markers CD25 and FoxP3, within the subpopulation of CD4+ T cells, was determined by flow cytometry according to manufacturer’s instructions . Data were acquired using a MACSQuant Analyzer 10 (Miltenyi) and analyzed with FlowJo .Animal care and treatment were carried out in accordance with Committee Stuttgart (Regierungspräsidium Stuttgart) guidelines on the use of experimental animals at the University of Stuttgart. Animal experiments were approved by Regierungspräsidium Stuttgart (permit no. 35-9185.81/0422). Oligomerized fusion proteins (1 mg/kg) were administered intraperitoneal (i.p.) to C57BL/6 wild type mice. After 3 days a second injection (1 mg/kg) was applied. After 7 days, spleens were extracted and splenocytes were isolated. Therefore, spleens were dissociated through a 40 µm cell strainer and collected in 10 ml MACS buffer . Splenocytes were centrifuged  and TNFR2 , followed by detection with appropriate fluorescence labeled antibodies. Nuclei were counterstained with DAPI. Stainings were analyzed on a Zeiss Axio Observer Spinning Disc microscope equipped with a Plan-Apochromat 63x/1.4 Oil DIC objective and an Axiocam 503 mono CCD camera. The following excitation lasers and emission filters were used: DAPI: 405 diode laser, 450/50 nm filter; GFP, 488 nm diode laser, 525/50 nm filter; RFP, 561 nm (RFP) diode laser, 600/50 nm filter. Z-stacks of tile regions containing 6 × 6 images were acquired and maximum intensity projections were calculated. Image processing was done in Zen blue 2.1 software .51. Nuclei were segmented via the DAPI staining and a 120 pixel wide ring mask was drawn around each nucleus representing the cell mask. TNF Vesicles were segmented under the cell mask using the A488 staining and unified for each cell. Mean intensity of the TNFR2 signal using the A546 staining was measured under the unified vesicles representing the grade of co-localized vesicles between TNFR2 (red) and TNF (green) per cell.Quantitative image analysis was done with CellProfiler version 2.2n independent experiments. Normal distribution was analyzed by Shapiro-Wilk normally test. Statistical analyses were performed by Student’s t-test or analysis of variance, followed by a post hoc Tukey’s range test. *P < 0.05  was considered significant.Data are presented as mean ± standard error of the mean (SEM) of Supplementary Data


HPV infections are associated with human cancers. Although three prophylactic vaccines have been approved to protect against HPV infections, the vaccines require cold-chain storage and may not be suitable for third world countries with less developed refrigeration facilities. We previously developed a bacteriophage L2 virus-like particle (VLP)-based candidate vaccine, which elicited broadly protective antibodies against diverse HPV types. Spray-drying of MS2-16L2 VLPs into a dry powder enhanced the stability of these VLPs. Building on these studies, we assessed the long-term stability and immunogenicity of the spray-dried VLPs. Mice immunized with a single dose of spray-dried MS2-16L2 VLPs, which had been stored for 14 months at room temperature (RT), were partially protected from challenge with a high dose of HPV16, one year after immunization. However, immunization with two doses of MS2-16L2 VLPs stored at RT for 34 months elicited high titer anti-HPV antibodies. More importantly, this group of mice showed significant protection from HPV16, 4 months after immunization. These results suggest that spray-dried MS2-16L2 VLPs retain their effectiveness after long-term storage at RT, and may be suitable in third world countries with less developed refrigeration facilities. This requirement comes with an additional challenge – the expense associated with refrigeration and the concern that the vaccines may be accidentally exposed to either freezing or warm conditions during storage or transportation, which may affect their efficacy. In fact, approximately 14–35% of all refrigerated vaccines  are accidentally exposed to freezing temperatures during cold-chain storage/transportation Warm temperatures can also cause problems. Exposure of the vaccines to >25 °C (room temperature) for more than 72 h may affect their efficacy We had previously reported the development of bacteriophage MS2 virus-like particles (VLPs), displaying epitope 17–31 from the minor capsid protein (L2) of HPV16, as a candidate HPV vaccine 22.1E. coli C41cells with pDSP62-16L2 plasmid. pDSP62-16L2 plasmid expresses the single-chain dimer of bacteriophage MS2 coat protein displaying L2 epitope 17–31 from HPV16 MS2-16L2 VLPs were made by transforming 2.2MS2-16L2 VLPs were formulated with a combination of excipients as follows: First, an excipient solution that consisted of 85.4% mannitol, 1.71% trehalose, 0.85% dextran, 7.85% L-leucine, and 4.27% inositol was prepared. VLPs were then diluted to 1.2 mg/ml using this excipient solution  and spray-dried using a Buchi Mini Spray Dryer B-290 with a standard two-fluid nozzle as previously described To address the effect of contaminating lipopolysaccharide (LPS) on the longevity of our VLPs, LPS was removed from VLPs using 1%  Triton X-114 as previously described 2.3For stability studies, MS2-16L2 VLPs were stored in air-tight containers at room temperature for 14–34 months after which they were reconstituted in sterile phosphate buffered saline buffer. An aliquot of the MS2-16L2 VLPs were observed under an electron microscope. Five μg of the MS2-16L2 VLPs stored for 14 months were used to immunize mice once  and 5 μg of VLPs stored for 34 months were mixed with alum hydroxide and were used to immunize mice twice with alum hydroxide. Another group of mice was immunized only once with 5 µg of LPS-free MS2-16L2 VLPs (not spray-dried) without alum hydroxide. Control mice were immunized with MS2 VLPs. All immunizations were done by intramuscular injection. Two weeks after the last immunization, sera were collected from mice immunized with VLPs stored for 34 months at room temperature and anti-HPV L2 IgG antibody titers were determined by peptide end-point dilution ELISA as previously published For protection studies, HPV pseudovirus 16 (representing HPV16), encapsidating a reporter plasmid (pClucf) encoding both luciferase and green fluorescence protein (GFP), were made, purified, and used to vaginally infect immunized mice as previously described 33.1We had previously devised a spray-dried candidate vaccine in which MS2-16L2 VLPs were formulated with a mixture of sugars  and an amino acid (L-leucine) into a dry powder. Spray-drying increased the long-term (7–14 months) stability of the VLPs at both room temperature and 37 °C without affecting its immunogenicity 4) against HPV L2 peptide representing epitope 17–31 from HPV16; antibody titers were similar to those from mice immunized with fresh liquid MS2-16L2 VLPs . Mice immunized with the LPS-free MS2-16L2 VLPs showed protection from vaginal infection with HPV PsV16 compared to control mice .Fig. 3Lo42/sr), computed by the software for control mice were less than the actual values. At a lower challenge dose, protection may have been significant. Unfortunately, the mice could only be challenged once with the same type of HPV PsV; there is little or no infectivity – due to immunity from prior challenge – if the same mice are re-challenged with the same PsV type.Current HPV vaccines are predicted to offer ~70–90% protection against cervical cancer and up to 86% protection against HPV-associated penile cancer. Nevertheless, the vaccines require continuous cold-chain storage and may not be suitable for third world countries with less-developed refrigeration facilities or in rural villages with no refrigeration facilities at all. Additionally, some vaccines in rural areas are ignorantly transported using frozen ice packs directly from freezers, which accidentally expose vaccines to freezing conditions, precipitating alum in the vaccines, thus reducing the efficacy of vaccines The level of protection can be enhanced further by immunizing with two doses of the spray-dried MS2-16L2 VLPs in alum hydroxide adjuvant. This view is supported by the fact that mice immunized with two doses of the spray-dried VLPs – stored at room temperature for 34 months – had similar antibody titers to mice immunized with two doses of fresh liquid MS2-16L2 VLPs B. MoreovSuccessful removal of LPS from our MS2-16L2 VLPs E Tumban and B Chackerian are inventors of L2-VLP related patent applications licensed to Agilvax Biotech. Interactions with Agilvax are managed by the University of New Mexico in accordance with its conflict of interest policies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


Tumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2’s E3 activity or RIPK1’s ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF.  •RIPK1 recruits the E3 ligase MIB2 to the TNF receptor signaling complex-I•Depletion of MIB2 sensitizes cells to RIPK1- and caspase-8-dependent cell death•MIB2-mediated ubiquitylation of RIPK1 represses its kinase activity and cell death•MIB2 acts as an additional survival checkpoint in TNF signaling Feltham et al. show that MIB2 directly ubiquitylates RIPK1 upon TNF stimulation, suppressing the cytotoxic potential of RIPK1 and acting as a checkpoint within the TNF signaling pathway. TNF functions as a master regulator of the cytokine network that coordinates defense of homeostasis by controlling inflammation, cell proliferation, differentiation, survival, and death . UbiquitcFlip.Binding of TNF to TNF-R1 results in the sequential formation of two signaling complexes . The rapThrough a process still ill defined, complex-I dissociates from the receptor, and RIPK1 together with TRADD associates with the adaptor protein FADD and pro-caspase-8 (casp-8) to form complex-II . The forBecause an inflammatory microenvironment can dictate the aggressiveness of certain tumors, and influence the response to therapy, a better understanding of the complex relationship between cell death and inflammation is critical. This is an important issue because its resolution, and putative therapeutic intervention, would allow the diversion of cancer-related inflammation into activation of cell death. Here, we report that Mind Bomb E3 Ub ligases contribute to the regulation of TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation.To elucidate what determines whether an inflammatory signal instructs either an innate immune response or triggers cell death, we undertook a proteomic-based approach using components of the TNF-R signaling complex (TNF-RSC) as affinity reagents. Proteins of the TNF-RSC were fused to 2x-HA tags and expressed in Flp-In T-REx-HEK293 cells that allow single-copy insertion of these components under the control of doxycycline (Dox). HA-tagged proteins were purified and the presence of co-purified proteins was determined via mass spectrometry. In addition to known constituents of the native TNF-RSC, we identified the RING-type Ub-E3 ligase Mind Bomb-2 (MIB2) as a RIPK1-interacting protein A. NotablConsistent with the notion that MIB2 is part of complex-I, and in agreement with a recent mass spectrometry study , we founMIB2, but not MIB1, dramatically sensitized 786-0 cells to treatment with TNF in the presence of TAK1i, in both short-term cell death experiments and long-term clonogenic survival assays  and to a lesser extent upon treatment with cycloheximide (CHX)  E and S2Ft manner E.Figure MIB2 significantly enhanced activation of caspase-8 and the sensitivity of cells to TNF-induced cell death in the presence of SM in multiple cell types  that activates TLR4 and drives TLR4-mediated and NF-κB-dependent production of TNF  oligos, induced cell death and loss of clonogenic growth potential   H–S3J. Fuh TNF/SM H. Taken h TNF/SM I.Figure MIB2 sensitized cells to caspase-8-mediated cell death, reconstitution with wild-type MIB2 suppressed the cytotoxic effects of TNF, as measured by caspase-8 maturation, generation of caspase activity (DEVDase assay), PARP cleavage, formation of complex-II, and viability assays  that abrogates its E3 ligase activity likewise failed to suppress TNF-induced cell death. Absence or mutational inactivation of MIB2 resulted in enhanced complex-II formation, caspase-8 activation, and cell death. Hence, physical association between MIB2 and RIPK1 is necessary but not sufficient to regulate RIPK1. This is evident because MIB2 RING finger mutants can still bind to RIPK1   residue(s) of RIPK1 that are ubiquitylated by MIB2, we used a semiquantitative mass spectrometry-based approach. In total, 14 K residues of RIPK1 were found to be modified by Ub C. These nd K634) D. K377 ond K634) , was notnd K634) E, this and K634) A. As expte RIPK1 A. Althouomplex-I F, this momplex-I G. This i binding B.K634R cells were considerably more sensitive than RIPK1WT cells when challenged with TNF/TAK1i  A–7D. GivNF/TAK1i . RIPK1K6PK1K377R A and S7B killing G. TogethL, are insufficient to prevent caspase-8-mediated cell death. Primarily this occurs via the induction of NF-κB and the upregulation of cFLIP, but recent evidence suggests that TAK1 and IKK can regulate TNF killing independent of their role in NF-κB activation , ubiquitylation of K604 and K634 is predicted to affect RIPK1 self-association, as these residues are positioned at the interface between two RIPK1 DDs. Moreover, K634 is located between RIPK1’s DD and FADD’s DD, which is predicted to directly affect the interaction if ubiquitylated.siMIB2 induces cell death is comparable with that triggered by TNF/SM  or engagement of multiple cytokine receptors .Further details and an outline of resources used in this work can be found in IkB-SR, Kym1, HT1080, MDA-MB-231, Jurkat, SWISS-3T3, HEK293T, and Flp-In-REx-HEK293 cells were cultured in DMEM; 786-0 cells were cultured in RPMI-1640 medium. Culture media were supplemented with 10% fetal bovine serum (GIBCO), penicillin, and streptomycin (GIBCO), and all cells were cultured at 37°C with 10% CO2 in a humidified incubator.HT1080MIB2 knockout clones or were further used to generate MIB1/2 knockout cells. Positive clones were characterized. Guide sequences are available upon request.Guide RNAs (gRNAs) were designed according to the Church lab  or ZhangImmuno-precipitation assays were performed as previously described . For all∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Statistical analysis was performed using GraphPad Prism version 6.0. Unless otherwise specified, data are presented as mean ± SEM. Comparisons were performed using a Student’s t test whose values are represented in the figures as


However, the negative regulation of TNF-α-mediated signaling remains undefined. Here we demonstrate the crucial role of miR-145 in the modulation of TNF-α-mediated signaling and cartilage matrix degradation. MicroRNA (miRNA) expression profiles of TNF-α-stimulated chondrocytes showed that miR-145 expression was rapidly downregulated by TNF-α. Moreover, miR-145 was directly repressed by p65 and was negatively correlated with TNF-α secretion during OA progression. Further, we found that miR-145 directly targeted mitogen-activated protein kinase kinase 4 (MKK4) and broadly restrained the production of several TNF-α-triggered matrix-degrading enzymes . Mechanistic studies unveiled that miR-145 negatively regulated TNF-α-mediated JNK and p38 activation, as well as the nuclear accumulation of p-c-Jun and p-ATF2, by inhibiting MKK4 phosphorylation, eventually resulting in the alteration of catabolic genes transcription. Indeed, p-ATF2 interacted with the promoter of Mmp-13, whereas p-c-Jun bound to promoters of Mmp-3 and Adamts-5. MKK4 was significantly elevated in OA cartilage. Eliminating MKK4 by short hairpin RNA resulted in obviously decreased matrix-degrading enzymes production, JNK and p38 inactivation, and an inhibition of cartilage degradation. On the contrary, MKK4 overexpression enhanced TNF-α-mediated signaling activation and transcription of downstream catabolic genes, and consequently worsened cartilage degradation. Moreover, intra-articular (IA) injection of miR-145 agonist to rat with surgery-induced OA alleviated cartilage destruction. Altogether, we elucidate a novel regulatory mechanism underlying TNF-α-triggered cartilage degradation and demonstrate the potential utility of miR-145 and MKK4 as therapy targets for OA.Cartilage dyshomeostasis contributes to osteoarthritis (OA) pathogenesis, and tumor necrosis factor (TNF)- In addition, as the main cytokines involved in OA pathogenesis, IL-1β is mainly associated with cartilage destruction, whereas TNF-α is implicated in driving of the inflammatory cascades during OA.9 Furthermore, chondrocytes from human OA cartilage present abnormal high expression of the p55 TNF-α receptor, which renders OA cartilage, particularly susceptible to TNF-α degradative stimuli. Meanwhile, OA cartilage produces more TNF-α and TNF angle convertase enzyme compared with normal cartilage.10 Therefore, targeting TNF-α signaling may be a powerful strategy to cure OA.It is known that inflammation presents cascade amplification,α have been intensively studied.12 In general, upon TNF-α binding to TNF receptor (TNFR), a conformational change promotes recruitment of cytosolic factors to the TNFR intracellular death domain (DD). Subsequently, TNFR-associated DD (TRADD) is recruited directly via a homotypic DD–DD interaction, and then the TRADD adaptor recruits a DD-containing kinase termed receptor interacting protein-1 (RIP1). RIP1 in turn recruits TNF receptor associated factor-2 (TRAF2) and/or TRAF5, as well as cellular inhibitor of apoptosis protein-1 (cIAP1) and cIAP2. These adaptors act in concert with RIP1 to engage the downstream phosphorylation cascades to activate NF-κB, JNK, and p38 pathways, which ultimately promote inflammation and transcriptional activation.15 However, the negative regulation of TNF-α-mediated signal transduction, especially the modulation of upstream molecules still remains largely unknown.Mechanisms that mediate the actions of TNF-19 MiRNAs regulate ~30% of human protein-coding genes, implying the essential role of miRNAs in controlling gene expression.22 Recent studies have illuminated several miRNAs related to cartilage breakdown triggered by IL-1β, including miR-140,23 miR-27b,24 miR-146a,26 miR-98,27 miR-558,28 and miR-30a,29 which commonly target a single molecule among MMPs and ADAMTS. However, at present, TNF-α-responsive miRNAs probably associated with OA development are poorly understood.MicroRNAs (miRNAs) are small noncoding RNAs of 19–24 nucleotides in length and exert their regulatory functions by mRNA degradation or translational inhibition.α-responsive miRNAs in chondrocytes and attempt to identify miRNAs intimately implicated in TNF-α-mediated signaling and investigate their potential roles in cartilage matrix degradation during OA pathogenesis. Our results show that miR-145 is sensitive to TNF-α, characterized by a rapid reduction upon TNF-α stimulation. In turn, miR-145 suppresses TNF-α-mediated JNK/c-Jun and p38/ATF2 activation and induction of MMP-3, MMP-13, and Adamts-5 by directly targeting mitogen-activated protein kinase kinase 4 (MKK4), and consequently attenuates cartilage matrix degradation in experimental OA.In this study, we explore TNF-β and TNF-α were strongly enriched compared with other cytokines  reduced C2C (a cleavage neoepitope of collagen II) concentration, cartilage destruction, and the upregulation of MMP-3, MMP-13, and Adamts-5 during experimental OA  extremely rescued the inhibitory effects of TNF-α on miR-145 expression  in the promoter region of Mmp-13, whereas c-Jun bound to promoters of Mmp-3 and Adamts-5 , or the entire CDS sequence of MKK4 (Len-MKK4) was generated. Indeed, Len-sh-MKK4 infection effectively depleted, whereas Len-MKK4 elevated MKK4, p-MKK4, and p-c-Jun  via IA injection of lentivirus carrying the GFP gene (Len-GFP), and found that Len-sh-MKK4 injection significantly reduced MKK4 in all joint tissues, except for meniscus  in consideration of the similar inflammatory conditions in patients with RA.TNF-α-mediated signaling activation and induction of cartilage matrix degradation mechanically through the MKK4-JNK/p38-c-Jun/ATF2 axis during OA pathogenesis, and demonstrate the potential utility of miR-145 and MKK4 as therapy targets for OA. To our knowledge, miR-145 represents the first identified TNF-α-reduced miRNA, which in turn serves as a negative regulator of TNF-α-mediated signaling activation and transcription.In conclusion, we elucidate a novel regulatory mechanism by discovering that miR-145 is a crucial negative regulator of TNF-n=10) who underwent total joint replacement. Undamaged areas in the same patient were sampled as the normal cartilage. Ethical approval was obtained from the Medical Ethics Committee of Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, and informed consent was obtained from all participants. All experiments involving rats were purchased from Shanghai SLAC Laboratory Animal Co. Ltd , and animal handling and experimental procedures were performed following the approval from the Institute of Health Sciences Institutional Animal Care and Use Committee. Experimental OA in 12-week-old SD rat  was induced by medial collateral ligament transection and DMM as described previously.38 SP600125  , SB203508  , CC-5013  , miR-145 agomir (50 μM), miR-145 antagomir (200 μM), and empty lentivirus (Len-C) or lentivirus  expressing sh-MKK4 (Len-sh-MKK4) or MKK4 (Len-MKK4) or the equivalent volume of vehicle (DMSO or PBS) were IA injected into the knee joints of recipient rats 1 week after the surgery (20 μl per joint per rat two times a week for 7 weeks) (n=6–10 per group). Rats were killed 8 weeks after the surgery, and samples of the knee joints were collected.Human OA cartilage was sampled from OA patients . After blocking with 1% bovine serum albumin (BSA), sections were incubated at 4 °C overnight with primary antibodies against MKK4 , phospho-MKK4 , phospho-c-Jun , phospho-ATF2 , MMP-3 , and MMP-13 .Human OA cartilage was fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 5 40 Cultured chondrocytes were maintained as a monolayer in DMEM/F12  with 10% fetal bovine serum (Gibco), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B under a humidified atmosphere of 5% CO2 in air at 37 °C. Recombinant TNF-α  was used to stimulate chondrocytes in complete medium. miRNA mimics or inhibitor  and siRNAs (GenePharma) were transfected with Lipofectamine 2000  according to the manufacturer’s instructions at a concentration of 40 nM. Sequences of miRNA mimics and inhibitor are shown in Rat articular chondrocytes were isolated from femoral heads, femoral condyles, and tibial plateaus of rats as described previously.α (10 ng/ml) for 24 h were determined by miRNA microarray analysis . Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter . Normalized data were further analyzed by two-tailed Student’s t-test. miRNAs with P<0.05 were considered differentially expressed  with high score (>100) were considered as candidate genes. The miRWalk database was further applied to exclude the genes that had been validated as targets of miR-145 in previous reports. The expression level of candidate genes in chondrocytes and cartilage were also evaluated by real-time PCR. Altogether, MKK4 was selected for further investigation.KpnI site of the GV268 vector using the following primers: 5′-ACGGGCCCTCTAGACTCGAGCATATAGCACCCCACACTG-3′ (sense) and 5′-TTAAACTTAAGCTTGGTACCGAGGTCCCAAGACCGCTTAC-3′ (antisense). The miR-145 inhibition plasmid was generated by the reverse complementary sequence of mature miR-145: 5′-AGGGATTCCTGGGAAAACTGGAC-3′, which was cloned into the BamHI/HindIII site of the GV249 vector. The following primers were used: 5′-AGCTAAAAAGTCCAGTTTTCCCAGGAATCCCTG-3′ (sense) and 5′-GATCCAGGGATTCCTGGGAAAACTGGACTTTTT-3′ (antisense). Fragment harboring the 3′-UTR of MKK4 containing the miR-145-binding sequence were cloned into the XbaI/XbaI site of the GV249 vector using the following primers: 5′-GATCGCCGTGTAATTCTAGAACATATTCATGAAATGTGG-3′ (sense) and 5′-CCGGCCGCCCCGACTCTAGAGCTGTGCAAGCTCTTCTC-3′ (antisense). For promoter reporter assay, fragment harboring the promoter of miR-145 containing the NF-κB-binding site (5′-GGGGATTCCTG-3′) was cloned into the XhoI/HindIII site of the basic vector  using the following primers: 5′-CCGCTCGAGCGGTCACGGTCCAGTTTTCCCAGGAATCC-3′ (sense) and 5′-CCCAAGCTTGGGCCATGACCTCAAGAACAGTATTTC-3′ (antisense). Site-specific mutant were generated by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit . All sequences of the amplified products were confirmed by DNA sequencing.The miR-145 expression plasmid was created as follows: the precursor sequence for miR-145 was cloned into the XhoI/Renilla luciferase plasmid (50 ng). For promoter reporter assay, SW1353 cells were transfected with a mixture of 200 ng luciferase vector (containing p145-wt or p145-NF-κB-mut) and 50 ng pRL-TK Renilla luciferase plasmid. Luciferase assays were performed with the dual-luciferase reporter assay system  according to the manufacturer’s instructions. Luminescent signals were quantified by a luminometer , and each value from the Renilla luciferase construct was normalized by firefly luciferase.All plasmids for transfection were prepared using the Qiagen Plasmid Purification Kit . The SW1353 cell line were co-transfected with miR-145 mimics or inhibitor (40 nM), luciferase constructs (described above) (200 ng), and pRL-TK (Promega) Chondrocytes were grown on a 20 mm glass bottom cell culture dish . Cells were fixed with 4% paraformaldehyde for 15 min, washed two times with PBS containing 0.05% Tween-20, permeabilized with 0.3% Triton X-100 for 5 min, and then blocked with 1% BSA for 30 min. Then, the cells were incubated at 4 °C overnight with the appropriate primary antibodies. Subsequently, the cells were washed with PBS and incubated for 1 h at room temperature with Alexa Fluor 488- or 594-conjugated goat anti-rabbit or donkey anti-mouse IgG secondary antibody (Invitrogen). 4′,6-Diamidino-2-phenylindole  was used for nuclear staining. Cells were visualized under a confocal microscope .RNAi was performed using siGENOME SMART pool siRNA  targeting rat MKK4 (gene ID 287398), HGK (gene ID 301363), p65 (gene ID 25716), Traf2 (gene ID 311786), and Tradd (gene ID 246756). The siRNA sequences are described in NotI/BamHI site of LV-5 (pEF-1a/GFP+Puro) using the following primers: 5′-TTAAGCTTATGGCGGCTCCGAGCC-3′ (sense) and 5′-CGGGATCCTCAGTCGACATACATGGGA-3′ (antisense).Chondrocytes were infected with Len-C, Len-sh-MKK4, and Len-MKK4 in combination with polybrene at the indicated MOI for 12 h, washed, and then maintained for 48–72 h before further analysis according to the manufacturer’s instructions. For Len-sh-MKK4 construction, LV3 (pGLVH1/GFP+Puro)  was used. The following primers were used: 5′-GATCCGGCAGATAATGGCAGTTAATTCAAGAGATTAACTGCCATTATCTGCCTTTTTTG-3′ (sense) and 5′-AATTCAAAAAAGGCAGATAATGGCAGTTAATCTCTTGAATTAACTGCCATTATCTGCCG-3′ (antisense). For Len-MKK4, the PCR products of MKK4 were cloned into the Chondrocyte apoptosis after transfection was determined by TUNEL assay using a kit  according to the manufacturer’s instructions. Specimens were visualized under a confocal microscope (Leica). Chondrocytes after transfection were also detected by Annexin V-FITC/propidium iodide double staining  with FACS analysis.−ΔΔCt) method. GAPDH and small nuclear RNA U6 were used as internal controls for cDNA and miRNA, respectively. The data in β-actin, due to the low uniformity of GAPDH among the different tissues. Each experiment was performed in triplicate. The primer sequences used in this study are summarized in Total RNA from tissues or cells was extracted using Trizol reagent (Invitrogen). First-strand cDNA was synthesized from 1 mg of total RNA by incubating for 1 h at 42 °C using the RevertAid Reverse Transcriptase  following oligonucleotide (dT) priming in accordance with the manufacturer’s instructions. qRT-PCR was performed by ViiATM 7 Real-Time PCR System (Life Technologies) using SYBR Premix Ex Taq . Data were analyzed using the comparison Ct . The membranes were blocked with 5% BSA and then incubated with specific antibodies overnight at 4 °C. The primary antibodies were from the following sources: Sox-9 , Col2a1 , MMP-3 , MMP-13 , Adamts-5 , COX-2 , JNK, phospho-JNK, Erk, phosphor-Erk, p38, phospho-p38, p65, phospho-p65, IκBα, phospho-c-Jun, phosphor-ATF2, MKK4, phospho-MKK4, HGK , and GAPDH . HRP-conjugated secondary antibodies  were used at a 1:1000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system  as recommended by the manufacturer.Cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors . Protein fractions were collected by centrifugation at 15 000 × μl of DNA samples with the primers shown in ChIP assay was performed using EZ ChIP Chromatin Immunoprecipitation Kit (Millipore) in accordance with the manufacturer’s instructions. Immunoprecipitation was carried out overnight with p65 , c-Jun , and ATF2  antibodies or normal rabbit IgG as a negative control. Protein A/G agarose was used to pull down the antigen–antibody compounds and then washed four times with washing buffers. The DNA–protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed with 2 α  or C2C  ELISA Kits (enzyme-linked immunosorbent assay). A standard curve was generated using known concentrations of the respective purified recombinant TNF-α or C2C.The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-t-test. P<0.05 was considered statistically significant.All statistical analyses were performed with SPSS 19.0 software . Data are presented as mean±S.D. 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