Begomovirus is essential for virus infection and is involved in multiple functions, such as virus movement and suppression of the host defence response. In this study, we reported that V2 encoded by the Tomato yellow leaf curl virus (TYLCV), which is one of the most devastating tomato-infecting begomoviruses, could interact with itself and a S71A mutation of V2 (V2S71A) abolished its self-interaction. Fluorescence results showed that V2 localized primarily in the cytoplasm and around the nucleus. Site-directed mutagenesis V2S71A had the similar subcellular localization, but V2S71A formed fewer large aggregates in the cytoplasm compared to wild-type V2, whereas the level of aggregates came to a similar after treatment with MG132, which indicates that the S71A mutation might affect 26S proteasome-mediated degradation of V2 aggregates. Meanwhile, heterologous expression of V2S71A from a Potato virus X vector induced mild symptoms compared to wild-type V2, delay of virus infection associated with mild symptoms was observed in plants inoculated with TYLCV-S71A, which indicates that the amino acid on position 71 is also involved in the pathogenicity of V2. To the best of our knowledge, this report is the first to state that the S71A mutation of V2 encoded by TYLCV affects the self-interaction, aggregate formation and pathogenicity of V2.The V2 protein encoded by  Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus of the family Geminiviridae, which is transmitted by whiteflies and causes one of the most devastating diseases in tomatoes worldwide4. TYLCV contains a single genome component with six open reading frames (ORFs) and an intergenic region (IR). Four ORFs  are located on the complementary strand, and the other two ORFs (V1 and V2) are located on the viral strand5. Replication-associated protein (Rep) encoded by C1, transcriptional activator protein (TrAP) encoded by C2 and replication enhancer protein (REn) encoded by C3 are involved in viral replication. C4 is likely involved in disease symptoms and viral movement. V1 is a capsid protein (CP) proposed to facilitate viral movement and assembly8.9, symptoms induction10, hypersensitive response (HR)11 and suppression of gene silencing12. For TYLCV V2, earlier studies have showed that V2 is involved in viral movement16. However, Hak et al. recently reported that this protein is essential for TYLCV infection, even though it is not directly involved in viral replication or movement17. While Moshe et al. found that V2 could bind genomic ssDNA and is occasionally co-localized with CP in large nuclear inclusions, it is likely to be involved in the CP-mediated DNA transport between the nucleus and cytoplasm, which indicates that V2 has a role as a DNA shuttling protein18. V2 was also reported to function as a viral suppressor of post-transcriptional gene silencing (PTGS) through interacting with slSGS3 and disturbing transportation of the RNA-silencing signal20. Recent research revealed that V2 could also suppress transcriptional gene silencing (TGS)21 in addition to PTGS. Moreover, Bar-Ziv et al. found that V2 could regulate plant defences through interacting with the tomato CYP1 protein, which is a member of the family of papain-like cysteine proteases22. Amin et al. found that expression of V2 via a Potato virus X (PVX) vector could induce severe symptoms in plants, which indicates V2 is involved in pathogenicity as an important symptom determinant23.Numerous studies had shown that begomovirus V2 proteins have diverse function associated with viral movement24. The TYLCV V2 protein was also reported to demonstrate aggregates formation in plants, and the formation and 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton, which indicated that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role in TYLCV infection18.Many plant and animal viruses induce the formation of insoluble aggregates/inclusion bodies inside infected cells. These structures usually contain viral and host proteins and can vary in location, size, content and biological function. For TYLCV, a recent report on CP protein showed that the progress of virus infection was accompanied by the formation of CP aggregates of increasing size, which might be involved in viral replication and assembly that finally affects the development of the viral diseaseRice black streaked dwarf virus (RBSDV) could interact with itself, which is associated with the formation of viroplasm-like structures to produce replication sites25. In Begomovirus, the self-interaction of Rep encoded by Abutilon mosaic virus (AMV) is involved in initiating the replication in the endonucleus26. The AL2 protein encoded by Tomato golden mosaic virus (TGMV) could interact with itself to form multimeric structures of AL2 in the nucleus and to activate transcription efficiently, whereas AL2 monomers present in the cytoplasm of infected cells, suppress local silencing by interacting with adenosine kinase ADK27. A recent study also found that the βC1 protein encoded by Tomato yellow leaf curl China betasatellite (TYLCCNB) lead to multimerisation by interacting with itself, which shows the multimers and self-interaction are related to begomovirus-like symptoms in N. benthamiana28.In many plant viruses, self-interaction of viral proteins is associated with their function. For examples, the P6 protein encoded by S6 of S71A) abolished its self-interaction, induced less cytoplasmic large aggregates formation and milder symptoms compared to wild-type V2. Additionally, our data indicate that an S71A mutation of V2 affects 26S proteasome-mediated degradation, which in turn altered the dynamics of aggregates formation. In summary, our results reveal a correlation between the amino acid on position 71, self-interaction of V2, aggregates formation and pathogenicity, which may have been largely unexplored in favour of determining the function of V2.Here, we show that the TYLCV V2 protein interacts with itself and that the amino acid on position 71 is crucial for the self-interaction. A substitution of serine for alanine at this position  contains 351 nucleotides (nt) (GenBank accession number: GU111505).Co-transformants of V2 cloned as a fusion with the GAL4 activation domain (AD-V2) and V2 as a fusion with the GAL4 DNA binding domain (BD-V2) were plated on different selective media to detect activation of the reporter genes HIS3 and ADE2. AD-V2 and BD-V2 were transformed into yeast (AH109) and the cells grew normally on SD/-Trp medium, which indicates that V2 non-specifically affected yeast-cell physiology. Yeast transformants containing AD-V2 and BD-V2 were able to grow and tended to appear blue on SD/-Ade/-His/-Leu/-Trp medium with X-α-gal, which was similar to the transformants containing AD-recT and BD-53 (positive control), whereas yeast transformants carrying AD-V2 with an empty vector (AD-V2 + BD) or BD-V2 with an empty vector (BD-V2 + AD) or AD-recT with BD-Lamp (negative control) were unable to grow on the synthetic medium lacking Ade, His, Leu and Trp Fig. .Figure 1N. benthamiana leaves. In this assay, V2 was fused to the N (YFPN) and C-terminal (YFPC) fragments of yellow fluorescent protein (YFP) respectively to generate the constructs pV2-YFPN and pV2-YFPC. Co-expression of pV2-YFPN and pV2-YFPC by agro-infiltration resulted in a clear YFP fluorescence signal in the cytoplasm of agro-infiltrated cells at 48 hours post-infiltration (hpi). No YFP fluorescence was observed when pV2-YFPN and pYFPC or pYFPN and pV2-YFPC were co-expressed  in agro-infiltrated sed Fig. , althougsed Fig. . These rN. benthamiana by agro-infiltration. Total protein extracts were immunoprecipitated by FLAG-T rap beads . The resulting precipitates were analysed by Western blot assays using an anti-YFP antibody . We found that V2-YFP was co-immunoprecipitated by FLAG-V2 but YFP was not co-immunoprecipitated by FLAG-V2  assay. In this assay, FLAG-tagged V2 (FLAG-V2) was co-expressed with YFP or YFP-tagged TYLCV V2 (V2-YFP) in -V2 Fig. . In addiThrough the above experiments, we confirmed that V2 could interact with itself, but determining which domain/motif/sites of V2 were essential for its self-interaction were still unknown.http://www.ebi.ac.uk/interpro/)  Fig. , and theo/) Fig. , which iG70A , V2S71A , and V2K73A , were constructed and their effects on the self-interaction were tested.To clarify the approximate sites in the above motif (amino acids 61-78) involved in the self-interaction, three mutants, including V2G70A or AD-V2K73A together with BD-V2 could grow well on selective medium, but transformants containing AD-V2S71A and BD-V2 could not grow on the SD/-His/-Leu/-Trp plates  were expressed in N. benthamiana by agro-infiltration . Compared to untreated leaves, inhibition of 26S proteasome led to an increase in the number of large aggregates in both V2-YFP and V2S71A-YFP infiltrated leaves. The number of large aggregates in V2-YFP and V2S71A-YFP infiltrated leaves reached the same level in MG132-treated leaves  was generated and the infectious clone(TYLCV-S71A) was constructed. Infectious clone of TYLCV-S71A and wild-type TYLCV were used to inoculate 71A Fig. . Real ti71A Fig. . These d34. Many viral proteins interact with viral factors and/or host factors to fulfil the functions required by the virus, including self-interaction. In this study, we found that the TYLCV V2 protein could interact with itself using the yeast two-hybrid system, and the self-interaction was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. A single amino acid mutation on position 71 of V2 abolished the self-interaction. To the best of our knowledge, this is the first report about self-interaction of V2 encoded by TYLCV.Numerous reports have shown that viral proteins are multifunctional during the processes of infection, replication, movement and pathogenicity19. A tomato homologue of the Arabidopsis SGS3 protein (slSGS3) was identified as the host-cell target of V2. The interaction between V2 and slSGS3 was involved in disturbing transportation of the RNA-silencing signal, which suggests there is a correlation between the V2–slSGS3 interaction in plants and the suppressor activity of V220. V2S71A retained the ability to interact with slSGS3  and Tomato Aspermy Virus (TAV) was demonstrated using BiFC, which proposed that 2b functions as a suppressor of RNA silencing in the form of a homodimer or higher order oligomer40. In Fijivirus, the self-interaction and high-order oligomers of P9-1 protein encoded by Rice black-streaked dwarf virus (RBSDV) were also found to be involved in the formation of viroplasm42. In Begomovirus, it was reported that the AL2 protein encoded by Tomato golden mosaic virus (TGMV) could interact with itself and form different oligomers with different subcellular localization and functions. Multimeric structures of AL2 were located in the nucleus to activate transcription, whereas monomers were located in the cytoplasm to suppress local silencing27. In this study, TYLCV V2 was demonstrated that it could interact with itself, which prompted us to investigate whether V2 was capable of forming multimeric structures and what were its biological functions. There was no evidence to support the presence of multimeric complexes of V2 was affected by the self-interaction or not yet, so further research on V2 multimerisation and associated biological functions should be addressed.Multimerisation of viral proteins play an important role in the life cycle of virus44. Whether there is a correlation between the phosphorylation on the serine on position 71 and the functions of V2 should be investigated in future research.Here, we investigated the self-interaction of V2 and found mutation of S71A on V2 abolished its self-interaction and affect V2 large aggregates formation and pathogenicity. The serine on position 71 of V2 is a potential phosphorylation site based on an online prediction. It was reported that the Ser188 phosphorylation of RhoA, a major regulator of the actin cytoskeleton, by cAMP and cyclic GMP-dependent kinases (PKA and PKG) could protect RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation, regulating RhoA expression and functions in vascular smooth muscle cells. The recent research on tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, also revealed that the phosphorylation of TH at its Ser19 also had effects on its proteasome-mediated degradationNicotiana benthamiana. The plants were grown in a growth chamber  with temperatures ranging from 26 °C  to 22 °C  for 1–1.5 months before infection with the viruses. After infiltration, the plants were kept under the same growth conditions.All agro-infiltration experiments were performed in 45 using corresponding primers G70A, V2S71A, V2K73A were synthesized  based on the sequence of wide-type of V2 and confirmed by sequencing , truncated mutants of V2-M1 (residues 1–103), V2-M2 (residues 1–78), V2-M3 (residues 1–60), V2-M4 (residues 61–116), V2-M5 (residues 61–103), V2-M6 (residues 79–116), V2-M7 (residues 79–103), V2-M8 (residues 61–78), V2-M9 (residues 104–116), and site-directed mutants V2S71A genes (BglII) were amplified using specific primers for PCR (Supplementary Table BamHI (compatible with BglII) site of p1300-YFP vector and fused in frame with YFP at the N-terminus to generate V2-YFP and V2S71A-YFP.To investigate the subcellular localization, the TYLCV V2 and V2S71A gene (BglII) were amplified using the primers listed in Supplemental Table BamHI site as a fusion with the N-terminal fragment of YFP, resulting in pV2-YFPN and pV2S71A-YFPN. The full-length coding sequence of V2 was cloned into the C-terminal fragment of YFP, resulting in pV2-YFPC.For the production of BiFC vectors, the full-length coding sequence of V2 and V2BglII) was amplified using specific primers listed in Supplementary Table BamHI site between the 35S promoter and the 35St in the pCambia1300 binary vector to generate FLAG-V2 for the future Co-IP experiment.FLAG tagged V2 , was generated. Then the full DNA-A of TYLCV-S71A was amplified using primers listed in Supplemental Table BamHI and SacI, then subcloned into the BamHI-SacI sites of the binary vector pBinPLUS to produce pBinPLUS-0.8A. The full-length of TYLCV-S71A was digested from pGEMT-1A with BamHI and inserted into pBinPLUS-0.8A at its unique BamHI site to yield clone pBinPLUS-1.8A. The infectious clone of wide-type TYLCV was constructed following similar procedure.For the construction of infectious clones of TYLCV containg V2S71A-YFP were introduced, respectively, into A. tumefaciens strain GV3101 through electroporation. Leaves of 4-week-old N. benthamiana plants were infiltrated with A. tumefaciens harbouring the described constructs. After 32 hpi, leaf explants were excised and YFP fluorescence was examined in epidermal cells using confocal microscopy (ZEIZZ LSM 710). The microscope was configured with a 458–515 nm dichroic mirror for dual excitation and a 488-nm beam splitter to help separate YFP fluorescence. The number of larger aggregates (>20 μm2) per image-area (4 mm2) were measured in 10 independent experiments.P1300-YFP, V2-YFP and V2in vivo treatment of N. benthamiana leaves, stock solutions were diluted in water to prepare solutions of 20 μM oryzalin, 10 μM latB and 50 μM MG132. The freshly prepared solutions were put in a syringe and used to treat the abaxial leaf side 24 h after agro-infiltration of V2-YFP or V2S71A-YFP. The leaves were examined under a microscope 15–20 h after inhibitor infiltration. Control infiltrations were performed using the same final concentration of DMSO without the drug.Oryzalin, latrunculin B (latB)  and MG132  were dissolved in dimethyl sulfoxide (DMSO) to prepare 100 mM stock solutions. For The yeast two-hybrid system was used to identify interactions between TYLCV V2 with itself, a truncated mutant or site-directed mutant.G70A, V2S71A, V2K73A) were linked to the B42 activation domain (AD)-containing vector expression vector then co-transformed into Saccharomyces cerevisiae AH109 with the LexA DNA binding domain (BD) containing V2 (BD-V2) to identify interactions. The plasmids BD-53 and AD-recT served as a positive control, plasmids BD-Lam and AD-recT were used as a negative control. Transformants grew at 30 °C for 72 h on SD/-His/-Leu/-Trp/synthetic medium and/or then transferred to the SD/-Ade/-His/-Leu/-Trp/medium containing X-α-gal to identify binding activity. Three independent experiments were performed to confirm the results.V2 and V2 truncated mutants  and site-directed substitution mutants  at 48 h after infiltration. YFP was observed under a mercury lamp light using a 488-nm excitation filter. Photographic images were prepared by using ZEN 2011SP1. Three independent experiments were performed to confirm the results.A. tumefaciens strain GV3101 carrying V2-YFP, V2S71A-YFP, FLAG-V2 and V2-YFP, FLAG-V2 and V2S71A-YFP or FLAG-V2 and p1300-YFP were co-infiltrated in N. benthamiana plants as descripted in Fig. 2, 2 mM EDTA, 2x EDTA-free proteinase inhibitor, 1 mM PMSF, 4 mM DTT, 1% glycerol, and 0.5% Triton-X100).40 hours after infiltration, the tobacco leaves were harvested and ground in liquid nitrogen. Proteins were extracted in extraction IP buffer (40 mM Tris-HCl at pH 7.5, 100 mM NaCl, 5 mM MgClNext, 0.4 g of each samples were ground into powder and the powder was mixed with 0.8 ml extraction buffer. The cell debris was pelleted by centrifugation at 12,000 g for 15 min. The supernatant was removed to incubate the samples with FLAG-conjugated beads. After 1 h of incubation at 4 °C, the beads were centrifuged and washed six times with IP buffer. Proteins were eluted by 20 µL of 2x SDS sample buffer and analysed by Western blot using a monoclonal anti-YFP antibody to survey whether V2-YFP can combine with FLAG-V2 or not. Meanwhile, after collecting the input sample and the loading proteins with 2x SDS buffer, both proteins were successfully detected using relative anti-YFP or anti-FLAG antibodies analysed with Western blot. Three independent experiments were performed to confirm the results.Supplementary information


Selaginella bryopteris water-soluble extract with and without dimethyl sulfoxide (Me2SO) for the efficient long-term storage of human umbilical cord blood- (hUCB-) derived MSCs. The cryopreservation experiment using the formulated freezing solution was further performed with hUCB MSCs in a controlled rate freezer. A significant increase in postthaw cell viability and cell attachment of MSCs was achieved with freezing medium containing Selaginella bryopteris water extract along with 10% Me2SO as compared to the freezing medium containing Me2SO (10% v/v) alone. Furthermore, the decreasing apoptotic events and reactive oxygen species production along with increasing expression of heat shock proteins also confirmed the beneficial effect of Selaginella bryopteris water extract. The beneficial effect of Selaginella bryopteris water extract was validated by its ability to render postpreservation high cell viability. In conclusion, the formulated freezing solution has been demonstrated to be effective for the standardization of cryopreservation protocol for hMSCs.The effective long-term cryopreservation of human mesenchymal stem cells (MSCs) is an essential prerequisite step and represents a critical approach for their sustained supply in basic research, regenerative medicine, and tissue engineering applications. Therefore, attempts have been made in the present investigation to formulate a freezing solution consisting of a combination of  In the last few decades, the advent of stem cell research has been a promising milestone in the field of cryobiology. Mesenchymal stem cells (MSCs) are specialized cells with a wide range of multiple therapeutic properties like anti-inflammatory, antiapoptotic, antifibrotic, and immunomodulatory along with tumor tropism and differentiation , 2. Thes2SO), glycerol, 1,2-propanediol, propylene glycol, polyvinylpyrolidone, and hydroxyethyl starch  octane (DABCO) were purchased from Invitrogen, USA. A LIVE/DEAD® Viability/Cytotoxicity Assay Kit was purchased from Thermo Fisher Scientific (USA). Triton X-100, ethanol, paraformaldehyde, 0.25% trypsin, and plastic wares were purchased from Himedia, India. All the primary antibodies were purchased from Abcam  and fluorescently tagged antibodies were purchased from BD Pharmingen .All the chemicals and reagents used in this study were purchased from Sigma-Aldrich Chemical  unless otherwise stated. Alpha-minimum essential medium  incubator for 24 h. The adherent mesenchymal stem cells (MSCs) were washed thoroughly with Dulbecco's phosphate buffer saline (DPBS) and then supplemented with freshly prepared expansion medium. The culture medium was changed twice in a week, and the cells were subcultured after reaching more than 80% of confluency.The mononuclear cells (MNCs) were isolated from hUCB by Ficoll-Paque density gradient centrifugation (700 ×g for 20 min). Then, the MNCs were resuspended in 5 cells) were stained with fluorescently tagged human monoclonal antibodies against CD34, CD45, CD73, CD90, and CD105. Then, the CD markers were analyzed using a flow cytometer .The specific surface antigens present on MSCs were characterized by a flow cytometer following the protocol earlier described by us , 19. TheSelaginella bryopteris were cleaned, dried in an oven at 40°C, and soaked overnight in minimal volume of water. Then, the leaves were again dried, washed twice, and further pulverized. Fine powder of the leaves (30 g) was added in 100 mL of autoclaved distilled water, and the mixture was continuously shaken for 4 h at 35°C. Further, the mixture was vortexed for 2 h and subjected to centrifugation at 3000 rpm for 1 h. Finally, the supernatant was filtered using a syringe filter (0.2 μm) and stored at −20°C till further experimentation.Leaves of Selaginella bryopteris aqueous extract (5% v/v) or Me2SO (10% v/v) and combination of both as a cryoprotectant in α-MEM medium supplemented with 15% FBS. The cryopreservation study was performed in a controlled-rate freezer following the cooling protocol as described previously [Selaginella bryopteris aqueous extract as its potential application in cryoprotection. For thawing of the cryopreserved MSCs, the cell suspension was placed in water bath (Lauda RM6) at 37–40°C followed by gradual dilution of cryoprotectant solution using prewarmed growth medium. Each batch of experiments was repeated at least three times.The culture medium of MSCs was replaced with different freezing medium formulations containing either eviously . After aeviously . After 7α-MEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution, and epidermal growth factor (10 ng/mL). The cultured MSCs were maintained in a humidified atmosphere at 37°C and 5% CO2 [The postthaw viability of cryopreserved MSCs was assayed by growing in d 5% CO2 . For a mμL) was added to each sample at room temperature, and after 10 min, cells were analyzed with a flow cytometer (FACS ARIA III) equipped with FACS Diva software [The efficiency of the freezing media was evaluated after 7 days of storage of MSCs in the liquid nitrogen. The cryopreserved MSCs were thawed and cell viability was assessed immediately after thawing. Cell viability was determined by using a LIVE/DEAD Viability/Cytotoxicity Assay Kit. The dye , grown over a coverslip, and fixed with 4% formaldehyde solution. Then, freshly prepared phalloidin Alexa Fluor 488 solution was added onto the coverslip to stain the F actin in MSCs. Finally, the cells were washed with mounting medium  and analyzed with confocal microscopy .μM) dye for 30 min at 37°C in the dark. After three times washing with PBS, cells were analyzed for ROS generation using a flow cytometer . At least 10,000 gated events were analyzed per sample by a flow cytometer.To evaluate the oxidative stress, ROS production in cryopreserved MSCs was assessed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) dye. Briefly, postthawed MSCs were allowed to be stained with DCFH-DA (20 μL of MTT solution (5 mg/mL) was added to each well followed by another 4 h of incubation at 37°C [To determine the postthaw cell viability of MSCs, the MTT -2,5-diphenyltetrazolium bromide) assay was carried out following the protocol described earlier by us . In brie at 37°C . After tμL PBS and allowed to be stained with 5 μL Annexin V-FITC and 5 μL propidium iodide for 1 h in the dark. Further, the apoptotic event in cryopreserved MSCs was quantified using a flow cytometer . The noncryopreserved MSCs were used as controls for the apoptosis study.An apoptosis study was carried out as a part of an evaluation of the quality of cryoprotectant solutions used for preserving MSCs . The evaμg) from each experimental group was electrophoresed and transferred to the PVDF membrane. The membranes were blocked with 5% nonskimmed milk and incubated with primary antibodies specific for Hsp70 (1 : 1000), Hsp90 (1 : 1000), and β-actin (1 : 10000). Then, the membranes were washed and incubated with secondary antibodies conjugated with horseradish peroxidase. The blots were developed and quantified using Super Signal West Fempto Chemiluminescent Substrate™  and Bio-Rad Versa Doc™ Imaging System 4000 .After cryopreservation in different cryoprotectant formulations, the postthawed MSCs were lysed with CelLytic M cell lysis reagent in the presence of 1X protease inhibitor cocktail. Then, the changes in expression of heat shock proteins Hsp70 and Hsp90 were studied by Western immunoblotting as described earlier . In briep < 0.05 was considered significant.The values are given as mean ± standard deviation of three independent experiments. The statistical differences among the experimental groups were assessed by one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison test using GraphPad Prism 5 (GraphPad). The characterization of purified population of MSCs was carried out by measuring positive expression of MSC-specific cell surface markers, namely, CD73, CD90, and CD105, and negative expression of CD34 and CD45. The hUCB-derived mononuclear cells were initially cultured for isolation of MSCs utilizing their plastic adherence property. The adhered cells showed prominent colonies within 5 to 7 days of culture. The flow cytometry data demonstrated the positive expression of CD73 (99.98%), CD90 (98.41%), and CD105 (99.96%) and negative expression of CD34 (0.0%) and CD45 (0.14%) markers . The finSelaginella bryopteris aqueous extract, Me2SO, and their combination on postthaw cell viability of MSCs, the MTT assay was performed and the results are highlighted in Selaginella bryopteris aqueous extract, Me2SO, and their combination. The cell viability of postthawed MSCs cryopreserved with Me2SO (10% v/v) was found to be 60 ± 2% when compared with that of noncryopreserved control MSCs. Moreover, MSCs cryopreserved with Selaginella bryopteris aqueous extract (5% v/v) have cell viability of 46 ± 5% when compared with noncryopreserved control MSCs. However, the combinatorial use of Selaginella bryopteris aqueous extract and Me2SO showed significantly increased cell viability of 75 ± 3% in postthawed MSCs  and Selaginella bryopteris aqueous extract (5% v/v). However, the events of apoptotic cell death in postthawed MSCs were comparatively lower during cryopreservation with Selaginella bryopteris (18.1%) aqueous extract when compared to Me2SO (22.94%) as shown in Selaginella bryopteris aqueous extract and Me2SO in combination as freezing medium demonstrated the significant reduced percentage of early-phase apoptosis (12.99%) among all the experimental groups. Furthermore, this reduction in apoptosis was also found comparable with that of noncryopreserved control MSCs (14.16%).Next, we investigated the apoptotic cell death in postthawed MSCs cryopreserved with tion Kit . A signiSelaginella bryopteris water extract. However, the maximum elevation in level of ROS was observed in postthawed MSCs cryopreserved with Me2SO when compared to noncryopreserved control cells. The combination of Selaginella bryopteris water extract and Me2SO significantly reduced the level of ROS production that was brought closer to the level of noncryopreserved control MSCs  was analyzed. It has been widely suggested that the overproduction of ROS is responsible for cellular death. In order to understand the precise mechanism of cell death and apoptosis, we hypothesized that the overproduction of ROS may be a major contributor of cell death. Interestingly, a dramatic increase in ROS was observed in postthawed MSCs cryopreserved with rol MSCs .Selaginella bryopteris water extract (5% v/v), Me2SO (10% v/v), and their combination. The noncryopreserved MSCs showed normal fibroblast-like cell morphology and intact organization of F actin after 48 h and 72 h of culture  in postthawed MSCs.In the present study, we analyzed the effectiveness of the cryopreservation procedure using the water-soluble extract of Selaginella bryopteris water extract with and without Me2SO as an effective cryoprotective solution for the cryopreservation of hUCB-derived MSCs. Thus, the present study deals with a reduction in Me2SO concentration as low as possible in freezing medium as well as an addition of natural osmolytes to avoid the cytotoxic effect of Me2SO and achieve improved recovery of MSCs after long-term storage. The overproduction of reactive oxygen species (ROS) during hypothermia and freezing is another important factor that causes free radical-mediated oxidative damage to macromolecules leading to the loss of a cell viability during and after freezing. An addition of suitable antioxidant in freezing medium is another important factor that helps to overcome the oxidative damage to the cells for maximum recovery during cryopreservation [Selaginella bryopteris acts as a potential antioxidant when added to the freezing medium as reported earlier [Selaginella bryopteris water extract was investigated to assess its effectiveness as a potential antioxidant in the present study which was found to exert a beneficial effect on the recovery of cryopreserved cells by efficiently scavenging freezing-induced elevated level of ROS in MSCs.Previously, we formulated a freezing medium containing trehalose, ectoine, and catalase for effective cryopreservation, and encouraging results were obtained with human umbilical cord blood (hUCB) mononuclear cells , 31. In  earlier . Thus, SSelaginella bryopteris water-soluble extract on cell viability of MSCs during cryopreservation. The cell viability of MSCs after cryopreservation was around 40% with Selaginella bryopteris water extract and 61% with Me2SO. The inclusion of Selaginella bryopteris water extract as cryoprotecting solution increases the viability and overall recovery of the hUCB MSCs. Thus, the cell viability data demonstrated the improved recovery of MSCs with Selaginella bryopteris water extract, suggesting a possibility of its use as an additive mixture during cryopreservation.In the first phase of the study, the major aim was to determine the effect of Selaginella bryopteris water extract in the presence and absence of Me2SO, and the data demonstrated that Selaginella bryopteris water extract protected the cells against freezing-induced apoptosis. Recent studies have reported an elevation in ROS like superoxide anion and hydrogen peroxide during the cryopreservation procedure [Selaginella bryopteris water extract in combination with Me2SO led to a significant reduction in ROS generation. Moreover, the prevention of ROS generation in cryopreserved MSCs by Selaginella bryopteris water extract indicates its involvement in the maintenance of mitochondrial health. The overproduction of ROS is a common phenomenon due to the occurrence of cryopreservation-induced stress. Thus, the study confirmed that the supplementation of Selaginella bryopteris water-soluble extract has a significant positive effect on mitochondrial health in postthawed MSCs.Next, we assessed the apoptotic and necrotic cell death of MSCs cryopreserved with rocedure . The eleSelaginella bryopteris water extract and Me2SO improved the cell viability and exhibited maximum retention of the cytoskeleton of cryopreserved and postthawed MSCs.The cytoskeleton such as filamentous actin performs multitude of functions such as maintenance of cell shape, muscle contraction, cell signalling regulating cell dynamics, and motility. Moreover, involvement of different proteins can also alter the organization and distribution of the cytoskeleton. The cytoskeleton integrity plays a major role in cell viability, proliferation, and differentiation. Loss of function of the cell membrane interferes with transport systems such as pH regulatory systems of the cell. Disruption of organelle membranes affects transport systems such as mitochondrial transport systems essential for oxidative phosphorylation, the major energy generating pathway . TherefoSelaginella bryopteris and associated protection of the cells against toxicity exerted by the use of a cryoprotective agent such as Me2SO as well as against the freezing-thawing injury. The results showed that the levels of Hsp70 and Hsp90 were significantly higher in MSCs cryopreserved in freezing medium containing Selaginella bryopteris water extract alone or in combination with Me2SO when compared with noncryopreserved MSCs. Thus, our data substantiate that Selaginella bryopteris aqueous extract improves stability and function of cryopreserved hUCB-derived MSCs.As a protective response to external stimuli, Hsps increase their expression, help to maintain the metabolic and structural integrity of the cells, and enable the cells to become more resistant to stress conditions –34. It i


Cirbp expression, the protective effect of CIRBP is lost. Here, we show that the TNF effect on Cirbp requires the non-canonical NF-κB signalling pathway. While a knock down of RelA does not alter the effects of TNF on Cirbp, a knock down of RelB represses this effect. In addition, the data indicate that p50 and p52 are required in the TNF induced inhibition of Cirbp. These results show that Cirbp expression in TNF treated cells is regulated via the non-canonical NF-κB pathway.The circadian clock is required for the rhythmic expression of a plethora of genes that orchestrate metabolism, sleep-wake behaviour and the immune response to pathogens. The cold-inducible RNA binding protein (CIRBP) is required for high amplitude expression of clock genes. Moreover, CIRBP protects the expression of clock genes from the inhibitory effects of tumour necrosis factor (TNF). However, since TNF represses  •Cirbp expression is inhibited by blocking IKKα or IKKβ.TNF effect on •Cirbp repression.Knockdown of RelB prevents TNF dependent •Cirbp expression in inflammation.Non-canonical NF-κB pathway seems to regulate  In mice it is well established that the response to various pathogens and to pro-inflammatory cytokines is under circadian control. Hence, the immune system of mice is especially sensitive to pathogens at the beginning of their active phase A recent study demonstrated that the cold-inducible RNA binding protein  interacts with transcripts associated in circadian behaviour and that CIRBP is required for high amplitude expression of clock genes Cirbp in fibroblasts and neuronal cells Cirbp knock out cells. On the other hand overexpression of CIRBP protects clock genes from the TNF effect Cirbp expression.Recently, we have demonstrated that TNF not only interferes with E-box mediated expression of clock genes, but also impairs the expression of In mammals, the NF-κB family comprises five different subunits, belonging to the Rel family, which can either hetero- or homodimerise in order to form transcriptionally active isoforms. These isoforms can have very different roles in the transcriptional activation or repression of inflammatory genes Cirbp expression. Our data point to the involvement of the non-canonical pathway in the TNF effect on Cirbp.Here, we show that inhibition of the NF-κB signalling prevents from the TNF mediated suppression of 22.12. For RT-qPCR analysis, cells were seed in triplicates in 12-well plates in a density of 1×105 per well. For Western Blots cells were seed in T25 flasks in a density of 1×106 per flask. Two days after seeding, cells were synchronized by serum deprivation (1% FCS). Whereas the NF-κB inhibitors IKK III  and IKK VII  were added immediately after serum deprivation, TNF (10ng/ml) (Peprotech) was added one hour later. The concentrations we used for the blockers of the NF-κB signalling pathway have already been described to be effective NIH3T3 fibroblasts (DSMZ), were grown in DMEM high glucose  medium (Gibco) supplemented with 10% FCS (PAN Biotech) and gentamicin . Cells were kept at 37 °C and 5% CO2.25). After 24 h they were transfected with DharmaFECT 1 transfection reagent (Dharmacon) and 25 nM of siRNA (Dharmacon) against p50, p52 or RelB. Gapdh and no target siRNA were used as positive and negative controls, respectively. The siRNA targeting RelA was obtained from QIAGEN. After transfection cells were incubated for 36 h for RNA analysis and 48 h for protein analysis.NIH3T3 cells and primary fibroblasts were seeded in 6-well plates  using qBase software Whole-cell RNA from cultured cells was extracted using NucleoSpin-RNA II kit RNA (Machery Nagel) according to protocol. Subsequently, 1 µg RNA was reverse-transcribed using random hexamers (Fermentas) and M-MuLV reverse transcriptase (Life Technologies). 20 ng of cDNA was amplified in a CFX384 detection system (Biorad) using the TaqMan precision PLUS Master mix (Primerdesign). The gene expression level was normalised to three housekeeping genes (2.4Cells were lysed with the IP lysis buffer (Pierce) as described in the protocol. Whole protein extracts (50 µg) in LDS sample buffer (Invitrogen) and DTT were applied on a NuPAGE 12% Bis-Tris-Gel (Invitrogen). The proteins were separated at constant 150 V in a MES SDS running buffer (Invitrogen). Subsequently, blotting on a PVDF membrane was performed in a full wet tank blot at 30 V. Membranes were incubated with a CIRBP rabbit polyclonal antibody recognising the C terminus of mouse CIRBP 2.5t-test. For grouped measurements with one variable for the selected genes, a one-way ANOVA was used, followed by a Bonferroni post-hoc test to decrease the likelihood of a rare event in multiple comparisons. P values less than 0.05 were considered significant, 0.01 in ANOVAs with post-hoc tests. Data are shown as mean +/− SEM of triplicate cultures.All experiments were performed in biological triplets and analysed with GraphPad Prism (GraphPad Software). To assess the statistical significance between single series of measurements we used the two-tailed Student’s 33.1CirbpCirbp by 29%. Treatment with IKK III (1 µM) reduced the extent of inhibition to 10%. The increase of IKK III concentrations to 4 µM enhanced basal expression of Cirbp and blocked the inhibitory effect of TNF. IKK VII in concentrations of 100 nM was sufficient to prevent any significant inhibitory effect of TNF on Cirbp. As described recently the effect of TNF on Cirbp expression is also seen at the protein level (Cirbp mRNA the expression of CIRBP protein was not enhanced when adding IKK III at higher concentrations (4 µM). Still, also at this concentration IKK III interfered with TNF mediated inhibition of CIRBP. The effects of IKK VII resembled those seen with the higher concentrations of IKK III, namely inhibition of basal expression of CIRBP and prevention of the inhibitory effect of TNF . When using higher TNF concentrations downregulation of clock genes was found to depend on TNF induced induction of Twist1 Cirbp expression to depend on NF-κB activation, it is interesting that also the upregulation of Twist1 by TNF is mediated through NF-κB signalling CIRBP has been shown to be required for high amplitude expression of clock genes Cirbp requires several components of the NF-κB signalling pathway, namely RelB, p50 and p52, while RelA seems not to be required for this effect. Thus, this process appears to be regulated through the non-canonical NF-κB pathway.In conclusion, we showed that TNF mediated downregulation of


Salvia miltiorrhiza Bunge. The mechanisms under its regulation of adhesion of molecular expression are explored. Here, the effect of DA on TNF-α-induced ICAM-1 expression was investigated in endothelial cells. TNF-α-induced ICAM-1 expression and subsequent adhesion of monocytes, as well as elevated reactive oxygen species (ROS) generation and NOX4 expression were all significantly reversed by DA, siNOX4 and NOX4 inhibitor GKT137831. Furthermore, TNF-α-induced ICAM-1 expression, which was increased via IKKβ/IκBα-mediated activation of NF-κB p65, was also inhibited by DA. Interestingly, NOX4 overexpression suppressed the ICAM-1 expression, and this finding may be ascribed to the activation of Nrf-2. Additionally, NF-κB inhibitor PDTC, siNOX4, or DA can decrease the TNF-α-induced ICAM-1 expression and suppress the adhesion of monocytes. In all, DA inhibited TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion to endothelial cells through the NOX4-dependent IKKβ/NF-κB pathway. Besides, NOX4 played dual role in regulating ICAM-1 expression via diverse signal pathway. This novel bioactivity will make DA a good candidate to be further explored for therapeutic or preventive application for atherosclerosis.ICAM-1 overexpression and subsequent adhesion of leukocytes to endothelial cells play critical roles in the early stage of atherosclerosis. Danshenol A (DA) is an abietane-type diterpenoid isolated from traditional Chinese herb  As an important proinflammatory cytokine, TNF-α activates endothelial cells at the site of inflammation, leading to the upregulation of endothelial cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1)2, intercellular adhesion molecule (ICAM-1)3, and endothelial leukocyte adhesion molecule-1 (E-selectin)4. The increased adhesion molecules mediate local leukocyte accumulation, adherence, and subsequent transmigration into subendothelial space, which is an early step in atherosclerosis5.Atherosclerosis is known as a chronic inflammatory disorder7. The expression of adhesion molecules is also widely considered to be activated in an oxidative stress scenario8. As such, we can reasonably speculate that ROS is responsible for the expression of TNF-α-induced adhesion molecules and the subsequent leukocyte accumulation. The multiple sources of ROS in vitro include NADPH oxidases, mitochondria, xanthine oxidase (XO), and lipoxygenase (LOX)13. In the past decades, the mitochondrion has always been considered as the main source of ROS in vascular cells14. Recently, the NADPH oxidase (NOXs) family, as an important source of ROS outside the mitochondria, has been implicated in the pathophysiology of vascular diseases16. Among the seven NOXs members , NOX1, NOX2, and NOX4 are the three main NADPH oxidases highly expressed in endothelial cells. At present, NOX2 and NOX4 are reported to be primarily responsible for ROS generation and have received much attention as regulators of vascular diseases, such as atherosclerosis and hypertension17. By contrast, recent findings suggested that NOX4 might also be vascular protective. NOX4 overexpression promotes proliferation and migration of endothelial cells but reduces serum-deprivation-induced apoptosis in human microvascular endothelial cells18. Furthermore, transgenic mice with endothelium-specific NOX4 overexpression exhibited enhanced vasodilation and reduced blood pressure19. More recently, endothelial NOX4 demonstrated protection of ApoE−/− mice from atherosclerotic lesions20.Increasing evidence has shown that TNF-α leads to oxidative stress by increasing reactive oxygen species (ROS) production23.Several lines of evidence have shown that the expression of TNF-α-induced molecules is regulated at the gene level by activation of transcription factors. These transcription factors, including Nrf-1, Nrf-2, and NF-κB p65, are redox sensitive and identified to be involved in the regulation of the gene expression of leukocyte-adhesion molecules and cytokinesSalvia miltiorrhiza Bunge (Danshen) is a traditional Chinese herb with a lot of properties that could improve our health. In pharmaceutical field, it has been a continuous source of therapeutic agents for cerebrovascular disorders while in food field, danshen is used as food additives to make herbal tea, soups, seasoner and beverages. Danshen is currently going through trials and may even become the first traditional Chinese remedy to gain approval by the U.S. Food and Drug Administration (FDA)24.25. Compared with well-known tanshinones such as tanshinone IIA, tanshinone I, and cryptotanshinone, its biological effects have been limited reported. Recently, studies focused on DA are paid more and more attentions. One paper reported that DA has a strong inhibitory effect on aldose reductase (AR)25. Also some study showed that DA has anti-inflammatory properties superior to those of tanshinone IIA26 hinting that DA has potential for anti-atherosclerosis. One early phase of atherosclerosis involves the recruitment of inflammatory cells from the circulation and their transendothelial migration. This process is predominantly mediated by cellular adhesion molecules27.Danshenol A Fig.  is an ab28. In the present study, we investigated the effects and potential mechanisms of DA on the TNF-α-induced ICAM-1 expression in human umbilical vein endothelial cells (HUVECs). We found that DA inhibited the TNF-α-induced ICAM-1 expression by suppressing the ROS-generating NOX4. Furthermore, NOX4 played a dual role in regulating the ICAM-1 expression induced by TNF-α.ICAM-1, one of the largest families of cell adhesion molecules plays an important role in the process of atherosclerosisWe first characterized the effects of TNF-α on ICAM-1 expression in endothelial cells. TNF-α significantly increased the ICAM-1 protein expression in a concentration- and time-dependent manner Fig. . The mRN2O2 production whereas DA had almost no effect on TNF-α-induced O2•− production  were studied. The TNF-α-induced ICAM-1 expression was inhibited by DA, NOX4 siRNA, or PDTC at both protein Fig.  and mRNAIn this study, we investigated the inhibitory effect of DA on TNF-α-induced ICAM-1 expression in endothelial cells. The results demonstrated the following: (1) DA inhibited TNF-α-induced ICAM-1 expression; (2) ROS-dependent NF-κB signaling played an important role in this process; and (3) NOX4 served a dual role in regulating the TNF-α-induced ICAM-1 expression.35, our results showed that TNF-α was a potent ICAM-1 inducer in endothelial cells at both protein and transcriptional levels. Immunofluorescence results suggested that TNF-α induced the membrane expression of ICAM-1. The inhibitory effect of anti-ICAM-1 on the TNF-α-induced adhesion of monocytes to endothelial cells showed that ICAM-1 plays an important role in cell adhesion.Consistent with previous reports2•−, H2O2, and peroxynitrite, act as signaling molecules to mediate various biological responses36. TNF-α-induced endothelial ROS production has been well established, but the detailed mechanisms remain unclear. In the present study, we found that TNF-α-induced ROS included both H2O2 and O2•−, which can be inhibited by both NAC and DA. Furthermore, DA showed no effect on DPPH free radicals. GSH comprises important intracellular antioxidants and antioxidative enzymes that help maintain the cellular redox homeostasis and protect cells from oxidative stress by directly scavenging ROS37. Interestingly, DA partly restored the TNF-α-induced decrease of GSH/GSSG ratio. Collectively, these data suggest that DA inhibited endothelial ROS formation. Accumulated data also suggest several sources of ROS involved in TNF-α-treated endothelial cells: NADPH oxidase, mitochondria, XO, and eNOS38. Under our experimental conditions, increased ROS was observed after TNF-α treatment, which was significantly suppressed by ROS scavenger NAC, NADPH oxidase inhibitor DPI, and DA. However, the inhibitors for the mitochondrial respiratory chain complex I-III, XO, and LOX showed no effects, suggesting that NADPH oxidase played an important role in this process. Considering that endothelial cells expressed several NOXs members39, the expression of NOX1, NOX2, and NOX4 was determined. The TNF-α-induced expression of both NOX2 and NOX4 contributed to the increased production of H2O2 and O2•−. However, DA significantly inhibited the expression of NOX4 without affecting NOX2. Thus, the inhibitory effect of DA on H2O2 and O2•− might be merely ascribed to its effect on NOX4. The small transmembrane protein p22phox serves a scaffold function for the maturation and folding of the catalytically active NOX protein through a proline-rich region40. TNF-α induced the membrane expression of p22phox, which was suppressed by DA. Collectively, these results suggest that TNF-α induced NOX4 activation in HUVECs.During ROS generation, a series of very small and highly reactive molecules, including hydroxyl radical (·OH), Oet al.41 reported that blockade of TNF-α-induced ROS by NOX4 siRNA suppressed the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion to human aortic endothelial cells. Also our previous study showed that NOX4 plays an important role in the process of atherosclerosis42. In the current research, we found that the knockdown of NOX4 by siRNA significantly inhibited the TNF-α-induced ROS production and ICAM-1 expression at both protein and mRNA levels, as well as the THP-1–HUVEC adhesions. Interestingly, the inducible expression of ICAM-1 in response to TNF-α was abolished in NOX4-overexpressed endothelial cells, although the generation of ROS was enhanced. Furthermore, TNF-α showed no obvious effect on Nrf-2 nuclear translocation in normal HUVECs but increased its nuclear translocation in NOX4-overexpressed HUVECs. This finding suggests that TNF-α activated the Nrf-2 pathways when NOX4 was overexpressed. Along with the role of NF-κB (discussed below), these results suggest that NOX4 played dual roles in the TNF-α-induced ICAM-1 expression mediated by NF-κB and Nrf-2.The role of NOX4 was further investigated. Jung 44. Most of these transcription factors are redox sensitive and can be activated by ROS45. Hence, the protein expression of NF-κB p65, Nrf-1, and Nrf-2 in the nucleus after TNF-α treatment was examined. Only the nuclear expression and nuclear translocation of NF-κB p65 were enhanced by TNF-α, and this upregulation was reversed by DA. IKKβ, whose activation results in IκBα phosphorylation, plays a critical role in the activation of NF-κB in the canonical pathway. The TNF-α-induced phosphorylation of both IKKβ and IκBα and the co-immunoprecipitation results suggest that their interactions were also enhanced. DA pretreatment partly reversed these alterations. Thus, these results suggest that TNF-α activated the NF-κB pathway by regulating IKKβ and IκBα. The inhibitory effect of DA suggests potential targets for this compound in the NF-κB pathway. The inhibitory effect of PDTC, an inhibitor of NF-κB, on the TNF-α-induced ICAM-1 expression and adhesion of THP-1 and HUVECs further confirmed the important role of NF-κB in the TNF-α-induced ICAM-1 expression.Transcription factors such as Nrf-1, Nrf-2, AP-1, and NF-κB have been reported to play important roles in TNF-α-induced ICAM-1 expressionIn conclusion, as depicted in Fig. 2-DA), N-acetyl cysteine (NAC), diphenyleneiodonium chloride (DPI), rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), antimycin A (AA), allopurinol (All), nordihydroguaiaretic acid (NDGA) were purchased from Sigma-Aldrich (USA). Lipofectamine TM 3000, dihydroethidium (DHE), and Amplex Red were purchased from Life Technology (USA). Antibodies for NOX1, NOX2, NOX4, p22-phox, Nrf-1, and Nrf-2, Na+/K+ ATPase as well as protein A/G PLUS-Agarose were purchased from Santa Cruz (USA). The antibody for ICAM-1 was purchased from Abcam Company (UK). Antibodies for p-NF-κB p65, NF-κB p65, p-IKKβ, IKKβ, p-IκBα, IκBα, GAPDH, and Histone H3 were purchased from Cell Signaling Technology (USA). BCA protein kits were purchased from Thermo Fisher (USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Aladdin (USA). siRNA for NOX4 was purchased from GenePharma Company (China). NOX4 overexpression plasmid was purchased from GeneChem Company (China). Primers and other materials for real-time PCR were purchased from Sangon Biotech (China) and TaKaRa Bio Group (Japan). P5 Primary Cell 4D-Nucleofector® X Kit L (24 RCT) was purchased from Lonza Company (Switzerland). GSH and GSSG assay kits were purchased from Beyotime Institute of Biotechnology (China). GKT137831 was purchased from BioChemPartner (China). FractionPREPTM Cell Fractionation Kit was purchased from BioVision (USA). All other chemicals were purchased from Sigma-Aldrich (USA).DA (>98%) was purchased from ChemFaces (China). Dimethyl sulfoxide (DMSO), 3--2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, 5-(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate  obtained from ATCC  was cultured in RPMI 1640 medium containing 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were seeded at 37 °C in a humidified atmosphere of 5% CO5 cells/well) and treated with DA in series of concentrations. Cell viability was measured after 24 h of incubation followed by addition of 20 µL of MTT (5 mg/mL). After 4 h, the MTT-containing medium was slightly removed, and DMSO (100 μL) was added to solubilize the formazan. Absorbance at 570 nm was recorded using a Spectra Max M5 Microplate Reader.HUVECs were planted into 96-well plates (1 × 10HUVECs were pretreated with DA (10 nM), anti-ICAM-1 Ab (10 μg/mL), or other inhibitors for 1 h followed by TNF-α (10 ng/mL) treatment for 24 h. THP-1 cells labeled with Hoechst 33342 were co-incubated with HUVECs for 3 h in the dark at 37 °C. Non-adherent THP-1 cells were washed with PBS. Images of the adherent cells were obtained under random fields of each well by using IN Cell Analyzer 2000.−ΔΔCt relative quantitative analysis to determine the expression differences.Total RNA was extracted using TRIzol reagent. Quantitative real-time PCR (RT-PCR) was performed using SYBR Green PCR reagents (Applied Biosystems) with forward and reverse primers for ICAM-1 and GAPDH. The specific primers for ICAM-1 were 5′-GGCTGGAGCTGTTTGAGAAC-3′ (forward) and 5′-ACTGTGGG GTTCAACCTCT G-3′ (reverse). For GAPDH, the primers were 5′-CGAGATCCC TCCAAAATCAA-3′ (forward) and 5′-TTCACACCCATGGACGAACAT-3′ (reverse). Briefly, 1 μL of cDNA from the RT reaction was added to 10 μL of the RT-PCR mixture containing 5 μL of Master Mix and 0.2 μM forward and reverse primers. The samples were incubated at 95 °C for 2 min, followed by 40 cycles of reaction at 95 °C for 15 s and combined annealing step at 60 °C for 1 min. The samples were assessed by 24 cells/well) were seeded on glass slides in 12-well plates. After TNF-α treatment (with and without DA pretreatment), the slides were fixed with 4% PFA for 30 min. The slides were then permeabilized with PBST (containing 0.1% Triton x-100 in PBS solution) and blocked with PBS (containing 4% BSA in PBS solution). After incubation with primary antibody (1:1000) and secondary antibody (1:2000), cells were stained with Hoechst 33342 and DiO-C5-3 in the dark for 30 min. Fluorescence was observed using IN Cell Analyzer 2000.HUVECs , NADPH oxidase inhibitor DPI (1 µM), mitochondrial complex I inhibitor Rot (20 µM), mitochondrial complex II inhibitor TTFA (10 µM), mitochondrial complex III inhibitor AA (5 µM), xanthine oxidase inhibitor All (10 µM), or lipoxygenase inhibitor NDGA (10 µM). After TNF-α treatment for 30 min, cells were washed with PBS and incubated with DHE (10 μM), Amplex Red (50 μM), or DCFH2-DA (10 μM) in the dark at 37 °C for 30 min. Fluorescence was measured using Spectra Max M5 Microplate Reader or via flow cytometry by using a FACSCantoTM system (BD Biosciences).The production of O6 cells/well) in six-well plates were incubated with TNF-α for 1 h after DA pretreatment. GSH and GSSG contents were determined using commercial kits in accordance with the manufacturers’ protocols.HUVECs  was incubated with methanol solution of DPPH (5.0 × 10−4 M) and kept in the dark for 20 min. The absorbance of the samples was measured using a Spectra Max M5 Microplate Reader at 517 nm.To evaluate the direct free radical scavenging effect of DA in a cell-free system, DPPH assay was performed as previously describedThe total proteins of the treated cells were collected. After quantitative determination of protein content, 30 μg of proteins was subjected to SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% nonfat milk in TBST  at room temperature for 1 h, the membranes were incubated with specific primary antibodies and secondary antibodies. Protein–antibody complexes were detected using an ECL Advanced Western Blot detection kit. The intensity of the bands was quantitated using Quantity One software (Bio-Rad).6/well cells were seeded in six-well plates overnight. Diluted siRNA and LipofectamineTM 3000 were mixed for 30 min at room temperature. Afterward, 200 μL of siRNA-lipofectamine complexes and 800 μL of Opti-MEM reduced serum medium were added to corresponding wells. The complexes were removed after incubation for 5 h, and cells were cultured with completed medium. The siRNA sequence for NOX4 was sense 5′-ACUAUGAUAUCUUCUGGUA-3′.Approximately 1.0 × 107 to 1 × 108 cells were resuspended in 100 μL of cDNA plus P5 primary cell solution and immediately electroporated in a 4D-Nucleofector X Kit L cuvette. Electroporated cells were transferred to six-well plates containing 2.5 mL of warm complete medium. Plates were cultured at 37 °C in a humidified atmosphere of 5% CO2. After 24 h, the medium was replaced with fresh medium at daily intervals.HUVECs were transfected by electroporation with plasmid encoding GFP-NOX4. For each transfection, cDNA (3 μg) was dissolved in 82 μL of nucleofector solution and then added with 18 μL of supplemented P5 primary cell solution. Approximately 1 × 10After determination of the protein concentrations, the cell extract was incubated with anti-IKKβ antibody (2 μg) for 2 h at 4 °C and then incubated with 20 μL of protein A/G plus-agarose beads overnight with constant shaking. Afterward, the beads were washed thrice with ice-cold radio immunoprecipitation assay (RIPA) buffer. The bound protein was extracted by adding 40 μL of 2 × SDS sample buffer and boiling for 5 min. The complexes were subjected to SDS-PAGE followed by Western blot.p < 0.05 was considered statistically significant.Data were expressed as means ± SD. Differences between groups were analyzed using Prism 5.0 , and statistical analysis was performed using one-way ANOVA, followed by Student–Newman–Keuls test. A value of


Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody  were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs  were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled  Tumor necrosis factor (TNF) is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but deleterious pro-inflammatory and cytotoxic functions during inflammation. TNF is a pro-inflammatory cytokine that represents a critical mediator of the autoimmune process, playing a key role in several inflammatory diseases, including rheumatoid arthritis (RA), ulcerative colitis, and Crohn’s disease. Increased understanding of the biological basis of autoimmunity has led to its identification as a major regulator of immune homeostasis, permitting the development of new TNF antagonists at the forefront of treatments for inflammatory conditions . TNF sigThe structure of the related lymphotoxin α  in complex with TNFR1 has shown that the trimeric cytokine binds three receptor molecules in a symmetrical way . The cytInhibition of the TNF has been achieved by the anti-TNF biologic etanercept, antibodies like infliximab and adalimumab, or with the antibody fragment certolizumab, used to treat autoimmune diseases. However, these therapies have many adverse effects, and a lot of patients do not respond or poorly respond to initial treatment, or lose their response with maintenance therapy due to immunogenicity or other causes , 7. An aWhile the antibody structure usually comprises a heavy chain combined with a light chain, camelids have antibodies that only consists of a heavy chain (HCAbs) . Variabl®), adalimumab (Humira®), and infliximab (Remicade®). However, these TNF antagonistic therapeutics act as bivalent molecules, resulting in higher avidity (We generated and isolated Nanobodies™ that were screened for inhibition of the interaction between TNF and the TNFR2. The TNF-specific Nanobodies™ were shown to inhibit the interaction between the cytokine and its receptor in enzyme-linked immunosorbent assay (ELISA). In addition, it is shown that these Nanobodies™ inhibit the TNF-induced necrosis effect in a cell-based assay. These bioassay data suggest that TNF:TNFR1 cascade is also inhibited, which is in accordance with previously published structural analysis of interaction between LTα and TNFR1 . Their p avidity . TherefoThe X-ray structures of three complexes of these Nanobodies™ and TNF make it possible to rationalize the impact of linker length on the potency of these TNF binders. This knowledge supports the rational design of the most optimal bivalent Nanobody™ constructs that demonstrate efficacy in the TNF transgenic mouse model of spontaneous arthritis.Escherichia coli using the method described by Marmenout et al.  as a recombinant protein in t et al. . Purifict et al. .Two llamas were immunized with TNF according to current animal welfare regulations, using the adjuvant Stimune . Two blood samples were collected from each animal as the source of B-cells. Total RNA was isolated according to the procedure described by Chomczynski and Sacchi . Random ® 96-well plate was coated with neutravidin and blocked, and biotinylated TNF was added to the wells. Phages were prepared as described by Marks et al. (®) for 30 min at room temperature, or by denaturation with acid (0.2 M glycine pH 2.5) for 20 min at room temperature. Two rounds of selections were performed.For the selection of Nanobodies™ against TNF, a Nunc Maxisorps et al.  and alloThe ability of the Nanobodies™ to inhibit receptor–ligand interaction was analysed in ELISA. A 96-well Maxisorp plate was coated overnight at 4°C with 2 µg/ml etanercept in PBS. Plates were blocked with 1% casein solution (in PBS) for 2 h at room temperature. Nanobody™ samples were preincubated for 30 min at room temperature with biotinylated TNF (200 pM). The mixtures were added to the plates and incubated for 1 h at room temperature. Biotinylated TNF was detected using Extravidin alkaline phosphatase  and pNPP  as substrate.4Ser(Gly)3Ser], 12GS [(Gly)3(Ser)]3, and 30GS [(Gly)4(Ser)]6 linker to connect the different Nanobodies™. The N-terminal VHH PCR fragment was digested with SfiI and BamHI, and the C-terminal VHH PCR fragment was digested with BamHI and BstEII. Ligations and transformations were carried out as described earlier.For construction of bivalent anti-TNF Nanobodies™, two separate PCR reactions were used to amplify the N-terminal and the C-terminal Nanobody™ subunits using oligo combinations containing sequences encoding a 9GS , which do not suffer from polymerase chain reaction artifacts during library construction. This confirms the occurrence of circulating B cells derived from an ancestor B-cell clone as a consequence of the in vivo maturation process.Recently, such variants of the anti-TNF VHH have also been identified E. coli expression vector with or without the carboxy-terminal c-MYC and/or hexa-histidine tags. After expression and purification, VHH were tested in the bioassay for their neutralizing capacity. Murine L929 cells expressing the mouse receptor were used for testing the VHH in combination with the human cytokine. By sensitizing the cells with actinomycin D, picomolar amounts of TNF were sufficient to induce the cytotoxic effect, which was taken as the assay read-out. All three VHHs were found to have low nanomolar potencies in line with the measured affinities for TNF , the potency of the Fab was approximately 30-fold lower.Because of the trimeric nature of TNF, we investigated whether the avid binding of a mAb such as infliximab contributes to its potency. Indeed, a considerable difference in potency was observed when the Fab fragment prepared by proteolytic digestion of infliximab was tested in the bioassay and compared with the intact antibody. The ICVHHs are strictly monomeric proteins and do not show any tendency to aggregate in multimers like single-chain variable fragments (scFv). Bivalent and bispecific formats were constructed by using linkers of variable length to fuse the VHHs. As a linker between the two VHHs, either a stretch of 9GS, 12GS, and 30GS sequence was used. Depending on the VHH and the length of the linker, a dramatic increase in potency was observed.KD) are 540 pM, 130 pM, and 1.5 nM for VHH#1, VHH#2, and VHH#3, respectively. In line with this, the IC50 values measured in the bioassay are in a similar range of between 240 pM to 1.5 nM  were determined by SEC. The trimeric TNF and the monovalent VHH molecules appear at elution times of 16.71 min (MW of around 50 kDa) and 15.68 min (MW 22.5 kDa), respectively  and TNFR2 (p75) and considering the high structural identity between TNF and LTα, a plausible model for the TNF/TNFR1 complex can be obtained by superimposing TNF onto LTα in its complex with the ectodomain of the TNFR1 p55 receptor (PDB entry 1TNR)  in the middle position fused with the 9 amino acid GS linker to both VHH#1 Nanobodies™, thereby bridging a distance of approximately 30 amino acids between the two TNF Nanobodies™, which according to the structural studies should be sufficient for enforcing intramolecular binding to the cytokine. For VHH#2 the albumin binding Nanobody™ was placed in the carboxy-terminal position thereby supporting the use of the smaller 9GS linker for directly connecting the two VHH#2 units that would be required for intramolecular binding. The potencies of the bivalent VHH#1 Nanobody™ (VHH#1-9GS-HSA VHH- 9GS-VHH#1) and the bivalent VHH#3 Nanobody™ (VHH#3-9GS-VHH#3-9GS-HSA VHH) in the cell-based potency assay were 22 and 18 pM, respectively. The potency of etanercept in this assay was 14 pM (data not shown). The amount of TNF used in the bioassay was 10 pM, which therefore represents the limit of sensitivity.p < 0.05) in the amelioration of clinical scores . Amelioration of arthritis by treatment of mice with etanercept at 10 or 30 mg/kg was also statistically significant in comparison to PBS treated group, whereas at lower doses (1 or 3 mg/kg) treatments with etanercept were no longer statistically significant. Complete attenuation of disease development was observed mice treated with 30 or 10 mg/kg of bivalent VHH#3, and in animals treated with 10 and 30 mg/kg of bivalent VHH#1. These results show that bivalent VHH#3 is improving clinical scores in all four doses tested ranging from 1 to 30 mg/kg. Bivalent VHH#3 is more potent than bivalent VHH#1 construct or etanercept. Bivalent VHH#1 improves the clinical scores at doses ranging from 3 to 30 mg/kg and is comparable to etanercept in terms of efficacy.The results of this study show that bivalent Nanobodies™ suppressed the development of arthritis in a dose-dependent fashion. In particular, administration either of bivalent VHH#3 at four different doses  or bivalent VHH#1 at three different doses  had a significant effect p < 0.0 in the ain vivo model  to achieve appropriate serum half-lives. Bivalent VHH#3 alone or in combination with VHH#1 Nanobody™ in a bispecific construct displayed the best binding to TNF. Their efficacy in vivo was found to be comparable to that of the receptor-based inhibitor TNFR-Fc etanercept. Topological considerations indicated that both the 9 amino acid linker of VHH#3-9GS-VHH#3-9GS-HSA and the ~30 amino acid (9GS-HSA-9GS) linker of VHH#1-9GS-HSA-9GS-VHH#1  provide distances compatible with the binding of both VHH#3 and VHH#1 modules to the same TNF molecule. This finding is key to explaining the excellent in vivo efficacy of these bivalent Nanobody™ constructs.Interestingly, a Tg197 transgenic human TNF mouse model for polyarthritis, confirmed This study was carried out in accordance with the recommendations of Ministry of Research, Belgium.in vitro and in vivo studies; writing of the manuscript. AD: performing structural studies. SS: crystallized the complexes, collected and integrated the X-ray data. ML: performing experimental work on nanobody production, formatting into multimeric constructs, and purification of complexes for structural studies. LA: bioassay measurements. TD: designing and supervising in vivo efficacy studies. CC: solved, refined the structures and wrote the paper. RL: interpretation of structural data and writing of sections on structural studies. KS: involved in experimental work and writing of the manuscript. CP: supervising the research, corresponding author. HH: inventor of and performed the first series of formatting experiments, initiated structural studies, and manuscript writing.EB: leading program, supervisor research EB, ML, TD, KS, CP, and HH: Ablynx employee during this research. AD, SS, LA, and CC: research paid in part by contract with Ablynx. RL: no conflict of interest.


By contrast, fish that consistently remained in the tank with the aggressor (Stay) showed significantly higher frequency of swimming in subordinate positions, at the top or the bottom of the water column, as well as sitting at the bottom. In addition, a corticotropin-releasing factor (CRF)-induced behavior, snap-shake, was also displayed in untreated fish during aggressive social interaction, and blocked by a CRF1 receptor antagonist. Especially prevalent among the Stay phenotype, snap-shake indicates indecision regarding escape-related behaviors. Snap-shake was also exhibited by fish of the Escape phenotype, showing a positive correlation with latency to escape. These results demonstrate adaptive responses to stress that reflect evolutionarily conserved stress neurocircuitry which may translate to psychological disorders and decision-making across vertebrate taxa.Socially stressful environments induce a phenotypic dichotomy of coping measures for populations in response to a dominant aggressor and given a route of egress. This submission- (Stay) or escape-oriented (Escape) dichotomy represents individual decision-making under the stressful influence of hostile social environments. We utilized the Stress-Alternatives Model (SAM) to explore behavioral factors which might predict behavioral phenotype in rainbow trout. The SAM is a compartmentalized tank, with smaller and larger trout separated by an opaque divider until social interaction, and another divider occluding a safety zone, accessible by way of an escape route only large enough for the smaller fish. We hypothesized that distinctive behavioral responses during the first social interaction would indicate a predisposition for one of the behavioral phenotypes in the subsequent interactions. Surprisingly, increased amount or intensity of aggression received had no significant effect on promoting escape in test fish. In fact, during the first day of interaction, fish that turned toward their larger opponent during attack eventually learned to escape. Escaping fish also learn to monitor the patrolling behavior of aggressors, and eventually escape primarily when they are not being observed. Escape  Adaptive behavioral responses to environmental stressors involve specific neurocircuitry-associated learning and conditioning, in all vertebrates, including rainbow trout   receptor antagonist, antalarmin. In mice, the anxiogenic drug yohimbine, a noradrenergic α2 receptor antagonist, blocks escape in most animals, which then remain submissively in the OF with an aggressor. These results suggest that multiple integrative stress neurocircuitries play a role in determining behavioral phenotype, and that these phenotypes are flexible  aspect of the SAM arena stimulates withdrawal from the OF  were reared in 50 foot long cement raceways under natural light conditions. The raceways were continuously supplied with aerated well water at 12°C. Fish were fed daily  at a rate of 1% body weight per day throughout the entire experiment. Large (350–570 g) adult hatchery brood-stock (N = 21) were used as the aggressive social stimulus (US) in this experiment. These fish were housed separately prior to experimentation, rotated and rested throughout the experiment to insure a high level of aggression toward the test fish. All experiments were conducted in a manner that minimized suffering and the number of animals used, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23), under a protocol approved by University of South Dakota IACUC.Social interactions using the fish version of the SAM arena were conducted at the Gavins Point National Fish Hatchery in Yankton, South Dakota. Rainbow trout (N = 80) were netted out of the group raceway and allowed to acclimate for 10 days in individual compartments of 75 gallon glass tanks with a flow-through water system and aeration. Each tank was individually lit and separated into four equal-sized compartments by the insertion of three opaque Plexiglas barriers. These UV lights were on at 6 a.m. off at 6 p.m. Fish were exposed to only ¼ of the tank during acclimation. The time for acclimation was chosen to stimulate territorial association with each compartment, while barriers inhibited social interaction between fish.Prior to training, small, juvenile (79–209 g) test fish (N = 30) that received a trace signal 15 s before social interaction with the possibility of escape through a hole . Fish choose, on their own, whether to escape (Escape) or remain submissively (Stay). Groups were designated a priori based on previous results, but were assembled only after behaving, to include: (1) trout that escape social interaction with a larger aggressor (Escape), and (2) those that remain submissively with the novel larger fish (Stay). During social interaction, all fish were exposed to ½ of the tank by divider removal. While all fish had the opportunity to escape using the hole, only those that did so had the possibility of exploring a 3rd compartment of the tank accessible only by the hole; these fish had no pre-exposure to the hole.Social Stress in the SAM arena Figure  typicallN = 8), or anesthetized (500 mg/l methane tricane sulfonate) and injected icv directly into the 3rd ventricle , 500 (N = 8), or 2,000 (N = 8) ng CRF   were observed through the complete set of SAM social interactions and treated with the CRF1 receptor antagonist antalarmin to determine whether endogenous CRF plays a role in Snap-Shake behavior in response to anxious indecision related to escape. For these trout, on day 4 of SAM social interactions, individuals from both escaping (N = 10) and non-escaping (N = 8) groups were orally administered the brain-permeant CRF1 receptor antagonist antalarmin (2 mg/Kg) in food, and assessed for snap-shake behavior.Another cohort of 32 fish was either untreated , and 15 s later, the barrier separating the large (US) and small fish was removed, along with the barrier that covered the escape hole (~3.8 cm dia.), making a neutral chamber available to the test fish. Fish were allowed to interact for 15 min, and latency to first attack, aggressive-, submissive-, and escape-related (retreat/explore) behaviors plus escape time (if applicable) were recorded. If the test fish did not escape within 15 min, the fish were separated, barriers re-inserted, water inflow was turned back on and the interaction was over. If the test fish did escape, the water was turned back on, the big fish moved back to his chamber, and the small fish was allowed to remain in the empty compartment for 5 min before being ushered back into the home chamber (by removing the divider). Pairings of CS:US occurred once daily over 7 days. Each test fish interacted with a novel large fish every day. Following 7 days of social interaction, on experimental day 8, fish were presented with the CS only (water off and barriers removed) for 15 min. No large fish was present. Behavior was recorded for 15 min. Immediately following this observation, test fish tissues were rapidly collected for analysis. Previous experiments also included aggression controls, in which pairs contested without access to an escape hole, as well as hole-only controls in which test fish were not paired with an aggressor, but did have access to the escape hole  recorded time of first attack and intensity of aggression by the larger fish, snap-shake anxious behavior, and position in water column of test fish, as well as aggressive, submissive, and retreat-explore (escape-related) behaviors of the test fish. Time of escape  was also recorded. Latency to escape is calculated from the time of first attack . The anxious behavior that we refer to as snap-shake is an integrated combination of biting motions, opercula flaring, and head shaking that occur together  using one-way ANOVA, including the analysis relative to behavior that preceded snap-shake. Linear regression analysis compared relationships between latency/escape learning curve and snap-shake performance and for number of escapes vs. accumulated social interaction in which the aggressor was not watching. For observed escapes, a curvilinear regression fit the data more precisely, and was therefore used. For all analyses, each animal provided only a singular datum. The data have been tested for the five assumptions of parametric statistics and transformed (log) when necessary. The data are analyzed both non-parametrically and using the parametric statistics previously mentioned, and for multiple comparisons using the Holm-Sidak method; when the statistical analyses match, as they do for the data reported herein, we report the parametric results (using tests listed above) without α adjustment to avoid increased Type II error  and Duncan's Multiple Range Test (to minimize Type II error).For all comparisons between groups (Escape vs. Stay), including analyses for intensity of attacks received, percent of snap-shake interactions, total submissive behavior exhibited over time, retreating/exploring over time, aggression over time, turning toward the attacker, movement directed at the escape hole, swimming at the bottom of the tank, or sitting at the bottom, Student's Rothman, . SignifiN = 10 for each group), with a third type of response not previously reported  = 1.945, p < 0.068] to have received more attacks than the Escape group  = 2.26, p < 0.037] intensity of attack than escaping animals  = 0.38, p > 0.71; Figure To examine the role of aggression in determining whether test fish escaped from the larger aggressor (Escape) or remained submissively (Stay) we examined attack intensity in three ways: (1) Average attack intensity received over each of 7 days of social interaction in the SAM, (2) the maximum attack intensity each day, averaged over 7 days, for Escape and Stay groups. Finally, we examined in the Escape group only, (3) the maximum intensity of attack received each day just prior (30 s) to escape. On the rare occasions when Escape group individuals did not escape, we used the maximum daily attack intensity (2) for comparison. For intensity of attacks received in a social interaction, we used the number of attacks occurring in any 3 s period as a gradient of intensity of attack that ranged from one (low intensity) to three (high intensity). When the intensity of attacks is averaged each day, and then over all 7 days, there is no broadly significant effect of aggression compelling escape behavior; and by contrast there is a trend for the Stay group  and Stay  groups after 7 days of social interaction, when limiting the analysis to behaviors on only the first day alone, submissive behaviors were by far the most prevalent, and occurred significantly more often than retreating, exploring, or aggressive behaviors  = 2.19, p < 0.042] more common for the Stay than the Escape group. In contrast, retreating/exploring  and aggressive  behaviors were similar between the two phenotypes.Although there was no clear behavioral Pavlovian conditioned response in trout that was coincident with the classically conditioned cortisol response  = 2.1, p < 0.05; Figure t(18) = 5.85, p < 0.001], before they actually escaped  = 3.24, p < 0.005] or at the surface , and to sit at the bottom  on the first day of social interaction, than fish that would eventually escape , were significantly more likely to turn toward their attacker on day one than were fish that reliably stayed with the novel aggressor on most of the 7 days of interaction [d Figure . Submiss Figures .Social interactions in the SAM apparatus produce dichotomous phenotypes that are not evident prior to SAM experiments   in both dorsal and ventral hippocampus , which reverses highly anxious submissive behaviors and allows for escape  block escape behavior, and promote submission  to escape (Carpenter et al., 1 receptor antagonism, is performed primarily after escape-related behaviors by submissive fish, and quantity of snap-shake performance is positively correlated in escaping trout with the latency to escape. Although there are significant barriers to learn escape, some trout make their escapes while the larger aggressive fish is not observing their movements, and clearly time their approach and execution for that moment. The fish and rodent versions of the SAM demonstrate a simple decision-making paradigm that describes an intensity gradient of anxiety and depressive behaviors for animals as evolutionarily distant as rainbow trout and mice (Carpenter et al., In conclusion, the two behavioral phenotypes expressed by a population of rainbow trout exhibit early behavioral indicators of the development of those phenotypes. Surprisingly, aggression Results from the new SAM conceptual model suggest that we can examine both susceptible and resilient individuals in real time, as well as begin to understand the behaviors and mechanisms that predict and produce these phenotypes. Behavioral outcomes in the SAM reflect changes in neurocircuitry and sensitivity to stressful sensory stimuli. While all individuals display submissive behavior during socially stressful interactions, susceptible individuals display specific types of submissive behavior, which are suggestive of withdrawal and behavioral inhibition. Resilient individuals on the other hand, face the social stress, learn from antagonistic social interactions and individuals, and use that information to escape from, and therefore ameliorate the socially stressful conditions. While increasing levels of anxiety result in a commensurate level of circumvention and indecision among resilient individuals, there appears to be a threshold level of anxiety that precludes resolving social stress through egress and commits an individual to submissive responses. While the data suggest interactive stress and arousal mechanisms (Carpenter et al., Participated in all aspects of the experimental, analysis, and writing tasks including substantial contributions to conception and design as well as acquisition, analysis, and interpretation of data, drafting, and revising the written text, final approval of the version to be published, and accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: TRS, TLS, RC, and CS. Participated in substantial contributions to conception and design as well as acquisition, analysis, and interpretation of data, revising the written text, final approval of the version to be published, and accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: JS and DA. Participated in substantial contributions in design as well as acquisition, analysis, and interpretation of data, drafting and revising the written text, final approval of the version to be published, and accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: SY, BM, and TO.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.