Additional T cell signaling cues triggered by co-stimulatory molecules and cytokines also affect TCR signaling duration, as well as accessory pathways that further shape a T cell response. Type 1 diabetes (T1D) is a T cell-driven autoimmune disease targeting the insulin producing β cells in the pancreas. Evidence indicates that dysregulated TCR signaling events in T1D impact the efficacy of central and peripheral tolerance-inducing mechanisms. In this review, we will discuss how the strength and nature of TCR signaling events influence the development of self-reactive T cells and drive the progression of T1D through effects on T cell gene expression, lineage commitment, and maintenance of pathogenic anti-self T cell effector function.T cell receptor (TCR) signaling influences multiple aspects of CD4 T1D is characterized by the destruction and/or dysregulation of the insulin producing β cells found in the pancreatic islets of Langerhans . Typicaltibodies , 23, 24.tibodies –18. For tibodies –30. CD4+subjects , 25–28. subjects . Moreoveantigens , 31, 32.uman T1D , 33, 34.+ (e.g. T helper 1 (Th1)) and CD8+ (e.g. type 1 CD8+ (Tc1)) Teff, marked by expression of IFNγ and other proinflammatory cytokines  expressed by pathogenic Teff . The TCRytokines , 51–53. ytokines –56. For ytokines , 58. Fursubjects . Interessubjects . These ssubjects . In addisubjects –66.+ T cells expressing the forkhead box P3 protein transcription factor  play a critical role in suppressing autoimmunity  , 94. Typvia selection events ongoing in the thymus (+ or CD4+ single positive thymocytes (SP), respectively. In the absence of TCR stimulation, DP undergo apoptosis. Since self-peptides drive positive selection, all functional TCR exhibit some degree of self-reactivity.TCR signaling plays a pinnacle role in regulating T cell homeostasis, activation, expansion and effector function upon recognition of cognate foreign- or self-antigens. The specificity and properties of the TCR repertoire are established e thymus –100. Pos+ SP expressing high affinity TCR for self-peptide, however, survive negative selection by differentiating into nFoxp3+Treg. SP that exhibit a low affinity for a TSA-derived peptide survive negative selection and egress from the thymus into the periphery (scid recipients reconstituted with T cells derived from transplanted thymic lobes of newborn but not 10 day or older NOD mice (Positively selected SP migrate from the thymic cortex into the medulla to undergo negative selection. The thymic medulla is populated by medullary TEC (mTEC), DC, and B cells which present peripheral tissue-specific antigens (TSA) –103. mTEeriphery . Noteworeriphery , 123. FoNOD mice . InteresNOD mice . The lac+Treg  versus B6 mice congenic for the H2g7 haplotype  and HLA-DQ2 (DQB1*02:01) –27. In Nimmunity , 143. No residue . This po alleles . Therefo+Treg. Dysregulation of these events can lead to an elevated peripheral frequency of autoreactive T cells expressing TCR with increased affinity, thereby enhancing susceptibility for pathogenic autoimmunity  not presented in the thymus, stimulate the expansion of diabetogenic Teff in NOD mice  the properties of the TCR repertoire expressed by T cells specific for self and foreign antigen, as well as 2) the development of protective nFoxp3immunity Figure 1NOD mice –148. IntNOD mice , 147.γ1, PI3K, MAPK, and Ca++ flux pathways , which serves to limit a given T cell response  is associated with increased susceptibility to T1D and other autoimmune diseases , 177–179ignaling . This anignaling . In thisignaling . Human Tignaling , 184. β ignaling . Interesl as Tfh .CTLA4 mRNA levels and altered mRNA splicing resulting in a soluble isoform of CTLA-4 (- T cells) is expected to readily promote expansion of Teff. Indeed, cancer immunotherapy trials have recently reported that CTLA-4 blockade induces T1D onset in some subjects   . Approac1 (PD-1) , 199. Sp+Treg stability  that regulates the CD8+ T cell response by influencing the magnitude of Ca++ signaling. ITK activation controls the expression of transcription factors such as Blimp-1, T-bet and IRF4 that are needed to generate terminally differentiated CD8+ Teff capable of cytotoxic function -specific OT-1 CD8+ T cells primed with high but not low affinity altered peptide ligands (APLs) induce diabetes upon transfer into transgenic mice expressing OVA by β cells. The pathogenicity of these CD8+ Teff in part correlates with upregulation of the integrin very late antigen-4 (VLA-4), which facilitates islet infiltration  infection . Sustainnditions , 188.A key exhaustion marker that has been associated with a Tex phenotype in multiple diseases is PD-1 , 272. Inmilieu during T1D limits the transcriptional development of terminally exhausted autoreactive T cells continues to be an area of study.The PD-1-PDL-1/2 axis is a prominent regulatory pathway in autoimmune diseases such as T1D , 277–281+Treg. On the other hand, dysregulation of signals 2 and/or 3 serve to modify signaling outcomes in peripheral β cell-specific T cells that favor differentiation and maintenance of pathogenic Teff and Tmem, while limiting terminal Tex cell development and a protective FOXP3+Treg response. The degree of dysregulation within the respective TCR signaling pathways may influence the heterogeneity observed in the pace of disease progression, the frequency of T cell infiltration of the islets, and the age of T1D onset in humans.The disease process of T1D is complex and heterogenous, influenced by genetic and ill-defined environmental factors. Multiple immune defects contribute to a failure of β cell-specific self-tolerance which impacts the function of various immune effector cells, including T cells. Genetic variants associated with increased T1D susceptibility have been linked to the generation of self-reactive TCR and dysregulated TCR signaling events. Evidence indicates that alterations in signals 1, 2, and 3 lead to impaired central and peripheral tolerance inducing mechanisms , and T32AI007273 (MC), and the American Diabetes Association 1-18-PDF-108 (MC).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.


To compare performance of new single-breath-hold 3D k-t BLAST cine b-SSFP sequence for left ventricular volume and mass evaluation using multiple-breath-hold 2D cine b-SSFP as a reference standard. We also compared time-efficiency of the two sequences calculating scan time and reporting time.On a commercially available 1,5 T MR scan , single-breath-hold 3D k-t BLAST cine b-SSFP sequence (3D-cine) and multiple-breath-hold 2D cine b-SSFP sequence (2D-cine) were performed in 46 patients referred to investigate different diseases. The global functional parameters, LV mass, scan time and report time were evaluated in each patient for both sequences. Differences between functional parameters and LV mass were made with a paired Student's T test; correlation between parameters was assessed with Pearson's correlation coefficient. A Bland-Altman analysis was used to investigate the limits of agreement between the measurements. Differences between time-efficiency related parameters were made with a paired Student's T test.Functional parameters and mass were significantly different in the two sequences (p < 0.05) but a strong correlation was found for LV ejection fraction (r = 0.96) and good correlation for other functional parameters (r between 0.83 and 0.93). Scan time was significantly lower for 3D sequence, report time was significantly higher for 3D sequence.3D k-t BLAST sequence can be used to assess EF in patients who have poor compliance in performing multiple apnoeas and in patients who are not able to remain in the scanner for a long time. Conversely report time is significantly higher for 3D sequence.


Together, these findings suggest that AR may decrease HCC progression through miR-325/ACP5 signaling and targeting the AR/miR-325/ACP5 signaling may help in the development of the novel therapies to better suppress the HCC progression.Hepatocellular carcinoma (HCC) is the most 5th commonly diagnosed and 2nd most lethal tumor in the world. The obvious gender advantage of HCC indicates that androgen receptor (AR) may play an important role in the tumor occurrence, develop and metastasis of HCC. Here we found that decreased AR could alter miR-325 to increase ACP5 expression in HCC cells, to increase HCC cells migration and invasion capacities. Mechanism dissection revealed that AR could regulate miR-325 expression through transcriptional regulation and miR-325 might directly target the 3'UTR of ACP5-mRNA to suppress its translation. The  Studies have shown that ACP5 is closely related to the cell invasion, disease progression and distant metastasis of many tumors, such as melanoma and breast cancer In this study, we demonstrated that decreased AR could downregulate the expression of miR-325 to promote the migration and invasion capacities of HCC by increasing ACP5 expression, thus we have discovered a new signaling pathway in the AR and HCC progression network, and at the same time, it provides the possibility of a new treatment method for inhibiting the progression of HCC.SK-HEP-1, 293T cell lines were purchased from ATCC. HA22T cell line was gifted from Prof. Yuh-Shan Jou, Academia Sinica, Taiwan. All cell lines were cultured in DMEM (Invitrogen) with 10% Fetal Bovine Serum, 1% penicillin/streptomycin and 1% glutamine and with a condition of a 5% (v/v) CO2 humidified incubator at 37 ºC.The pLKO.1-shAR, pLKO.1-shACP5, pLKO.1-shSMAD3 plasmids were transfected into 293T cells and cultured in the virus room. After 48 hours of transfection, the virus solution was collected and used immediately or frozen in a -80 ºC refrigerator for subsequent use.pLKO.1-shAR sequence, Forward: 5'-CCGGGAGCGTGGACTTTCCGGAAATGGATCCATTTCCGGAAAGTCCACGCTCTTTTTG-3'; Reverse: 5'-AATTCAAAAAGAGCGTGGACTTTCCGGAAATGGATCCATTTCCGGAAAGTCCACGCTC-3'.pLKO.1-shACP5 sequence, Forward: 5'-CCGGCCTCGGGCAAGTCCCTCTTTAGGATCCTAAAGAGGGACTTGCCCGAGGTTTTTG-3'; Reverse: 5'-AATTCAAAAACCTCGGGCAAGTCCCTCTTTAGGATCCTAAAGAGGGACTTGCCCGAGG-3'.pLKO.1-shSMAD3 sequence, Forward: 5'-CCGGGCCTCAGTGACAGCGCTATTTGGATCCAAATAGCGCTGTCACTGAGGCTTTTTG-3'; Reverse: 5'-AATTCAAAAAGCCTCAGTGACAGCGCTATTTGGATCCAAATAGCGCTGTCACTGAGGC-3'.The plasmids sequences used were as follows:Trizol reagent  was used to extract total RNA, and 2ug of the total RNA was used to reverse transcribe into cDNA by using Superscript III transcriptase . Relative RNA expression was conducted by Quantitative real-time PCR (qRT-PCR) assay by using a Bio-Rad CFX96 system with SYBR green reagent and the results were normalized by GAPDH.AR primer, Forward: 5'-CCAGGGACCATGTTTTGCC-3'; Reverse: 5'-CGAAGACGACAAGATGGACAA-3'.ACP5 primer, Forward: 5'-TGAGGACGTATTCTCTGACCG-3'; Reverse: 5'-CACATTGGTCTGTGGGATCTTG-3'.GAPDH primer, Forward: 5'-GGAGCGAGATCCCTCCAAAAT-3'; Reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3'.hsa-miR-194-5p primer: TGTAACAGCAACTCCATGTGGA.has-miR-512-5p primer: CACTCAGCCTTGAGGGCACTTTC.has-miR-6889-3p primer: TCTGTGCCCCTACTTCCCAG.has-miR-1227-3p primer: CGTGCCACCCTTTTCCCCAG.has-miR-6529-3p primer: CCTGTGCCTTTTACTTCTTTAA.has-miR-4266 primer: CTAGGAGGCCTTGGCC.has-miR-200a primer: CATCTTACCGGACAGTGCTGGA.has-miR-325 primer: CCTAGTAGGTGTCCAGTAAGTGT.has-miR-182 primer: TTTGGCAATGGTAGAACTCACACT.has-miR-370 primer: CAGGTCACGTCTCTGCAGTTAC.The primer sequences used were as follows:RIPA lysate was used to lyse the collected cells, and 30 ug protein was electrophoresed in 10% SDS/PAGE gel and then transferred onto PVDF membranes. After that, specific primary antibodies were used to incubate the relative bands for overnight in a 4 ºC room and then incubated with the secondary antibodies for 1 hour and visualized with ECL system (Thermo Fisher Scientific). Alpha-Tubulin (TU-02), GAPDH (6C5), AR (N-20), MMP1 (3B6), MAOA (G-10), SMAD3 (38-Q) and KIF4A (E8) antibodies were purchased from Santa Cruz Biotechnology. RPS4X (ab211427) antibody was purchased from Abcam. ACP5 (DF6989) antibody was purchased from Affinity biosciences. For protein stability assay, SK-HEP-1 cells were transfected with control or AR specific cDNA. After three days, cells were treated with 10 μg/ml of cycloheximide (CHX) for indicated times. ACP5 protein was detected by western blot.5 cells/well were seeded into upper chamber coated with diluted Matrigel  with serum-free medium and 750 μL media with 10% FBS/well was added into lower chambers for incubation for 36-48 hours. The invaded cells were fixed by methanol and stained by 0.5% (w/v) crystal violet. Each sample was run in triplicate and repeated multiple times.The invasion experiment is completed with a 24-well plate and an 8 μm chamber system (Corning Life Science). 1×10Cells were seeded into 35-mm plates until they are confluent, and the plates were scraped using a sterile pipette tip to create a wound through the confluent monolayer, then cultured in serum-free medium for SK-HEP-1 with 12 hours and HA22T with 24 hours and photographed at 0 and 12/24 hours, respectively. Measure the distance of wound migration for further analysis.350 bp fragment of human ACP5 3'UTR with wild or mutant miRNA-responsive elements was cloned into psiCHECK2 vector  downstream of the Renilla luciferase ORF. Cells were plated in 24-well plates and transfected the cDNA with Lipofectamine (Invitrogen) as the manufacturer's instruction. Dual-Luciferase Assay (Promega) was used to calculate luciferase activity according to the manufacturer's manual after 48 hours.6 HA22T plko+vector control (Group 1), HA22T shAR +vector control (Group 2), HA22T shAR+oemiR-325(Group 3), all cells were injected in situ under the liver capsule of the left extrahepatic lobe of the mouse. The IVIS system was used to detect tumor growth once a week. The study was carried out under the approval of the ethics committee of Xiangya Hospital Central South University.24 6-weeks old nude mouse were divided into 3 groups: 1×10All statistical was analyzed by SPSS 22.0 system . The data values were presented as the mean ± SD. Differences in mean values between two groups were analyzed by two-tailed Student's t test and the mean values of more than two groups were compared with one-way ANOVA. p≤0.05 was considered as statistically significant.Figure ) and overexpressed AR in HCC SK-HEP-1 cells , and then we checked AR's effect on HCC progression through wound-healing migration assay and transwell invasion assay. The results showed that knocking down AR could increase HCC cells migration and invasion capacities , and overexpressing AR led to decrease HCC cells migration and invasion capacity .To study the potential effect of AR on HCC cells, we first knocked down AR in HCC HA22T cells . Then we knocked down/overexpressed ACP5 in HCC cells and checked the migration and invasion capacities in these cells, the results showed that knock down ACP5 could decrease HCC cells migration and invasion capacities and oeACP5 could increase HCC cells migration and invasion capacities . Next we conducted reverse assay to checked if shACP5 could really reverse shAR's function to HCC cells, the results showed that shACP5 could partly reverse shAR's function to HCC cells , and oeACP5 could partly revers AR's function to HCC cells . For SMAD3, when we shAR/shSMAD3 in HCC cells, the invasion capacity could not be reversed . Moreover, when we manipulate AR, the mRNA level of ACP5 did not have a consistent change with AR level .To dissect the mechanism of how AR can decrease HCC cells migration and invasion capacities, we first searched some published papers that were related to cancer progression, and from which, we selected MMP1, MAOA, RPS4X, ACP5, SMAD3 and KIF4A genes, and then we conducted western blot assay to check these genes expressions when knocking down/overexpressing AR in HCC cells. The results showed that ACP5 and SMAD3 might be potential candidate of AR, for their expression was decreased/increased when overexpressing/knocking down AR in HCC cells . Then we focused on miRNAs, for many papers showed that miRNAs that can regulate gene expression by binding to the 3'UTR of specific target mRNAs to inhibit translation or to induce degradation. Firstly, we selected some potential miRNAs that can regulate ACP5 expression through online databases . Then we checked these miRNAs expressions when knock down/overexpressing AR in HCC cells, and the results showed that miR-325 and miR-370 might be the potential candidate to regulate ACP5 expression . Next, we added miR-325 inhibitor/overexpressing miR-325 in HCC cells and checked the migration and invasion capacities of them, the results showed that adding miR-325 inhibitor could increase HCC cells migration and invasion capacities and increase ACP5 expression at the same time , and oemiR-325 could decrease HCC cells migration and invasion capacities and decrease ACP5 expression at the same time . For miR-370, when we added miR-370 inhibitor into HA22T cell, the ACP5 expression was not increased , we suspect it may could not regulate ACP5 expression in HCC cells.To dissect the mechanism of how AR can decrease ACP5 expression, we first checked ACP5 stability after overexpressing AR in HCC cells, and the result showed the protein stability was not changed significantly , while adding miR-325 inhibitor could partly reverse oeAR's function to HCC cells , which indicated that miR-325 could be regulated by AR to regulate HCC progression.Next, we conducted the reverse assay to check if miR-325 inhibitor/oemiR-325 could reverse oeAR/shAR's function to HCC cells. The results showed that oemiR-325 could partly reverse shAR's function to HCC cells . We then applied the chromatin immunoprecipitation (ChIP) assay to verify their binding to AR in HCC cells, and results revealed that AR could bind to the ARE-2/3 region .To further dissect the molecular mechanism of how AR can regulate the miR-325 expression at the transcriptional level, we searched for the potential AREs on the 2 Kb region of the miR-325 promoter using JASPAR database, and results revealed that 4 putative AREs were located on the regions  . As expected, the luciferase assay results revealed that knocking down AR significantly decreased luciferase activity in HA22T cells transfected with wild type ARE, but not in the cells with mutant ARE . When we replaced the HA22T cells with the SK-HEP-1 cells, adding AR significantly increased the luciferase activity in the SK-HEP-1 cells transfected with wild type ARE, but not in the cells with mutant ARE .We then constructed the miR-325 promoter luciferase reporter construct by inserting a 1 kb 5′ promoter region of miR-325 into the pGL3 luciferase backbone as well as a version with the mutated ARE . Results from the luciferase assay revealed that addition miR-325 inhibitor increased luciferase activity in HA22T cells, while addition of miR-325 decreased luciferase activity in SK-HEP-1 cells transfected with wild type ACP5 3'UTR but not the mutant ACP5 3'UTR , suggesting that miR-325 can directly target the 3'UTR of ACP5-mRNA to suppress its protein expression.To dissect the mechanism of how AR-miR-325 axis can alter the ACP5 expression at the molecular level, we first searched potential miR-325 targeting site located on the 3'UTR of ACP5-mRNA. We then applied the reporter assay with the psiCHECK2 vector carrying the wide-type 3'UTR and a deletion mutant without the miRNA-target site  to analyze ACP5 expression in normal liver tissues and HCC samples in TCGA database. The results showed that ACP5 expression was higher in primary tumor samples than in normal samples . Then we used Protein atlas website (https://www.proteinatlas.org/) to analyze IHC data of ACP5 expression in HCC samples, and the IHC data showed that ACP5 expression was higher in primary tumor samples than in normal samples . Next, we used GEPIA website to analyze the correlation between AR and ACP5, the result showed that the expression of ACP5 had negative correlation with AR . Moreover, we analyzed the ACP5 expression in metastasis and different tumor stage in TCGA database, the results showed that ACP5 expression was higher in M1 (with metastasis) tumor than in M0 (no metastasis) tumor , and ACP5 expression was higher in stage IV tumor than in stage I and stage III tumors .To link the above in vitro studies to the clinical significance, we first knocked down AR in HA22T cells and got the stable transfected cells, then we transfected the HA22T shAR cells with luciferase reporter gene [for in vivo imaging system (IVIS)] to detect tumor progression inside mouse and then transfected the HA22T shAR cells with vector control or oemiR-325 plasmids. The HCC cells were divided into three groups, HA22T plko+vector control (Group 1), HA22T shAR +vector control (Group 2), HA22T shAR+oemiR-325 (Group 3). Tumors growth and metastasis were monitored weekly via IVIS analysis.To link our in vivo function of knocking down AR , we also count the mice with metastasis tumors, the results also showed that knocking down AR could promoter tumor metastasis , and we also counted the metastatic foci in each mice, the results showed the same tendency with Figure Figure ), what's more, the IHC data of ACP5 expression also confirmed our conclusions .Our results revealed that xenografts of HCC cells transfected with shAR plasmid (Group 2) had more progression than control group (Group 1), and oemiR-325 could partly reverse Figure 7 suggests that AR/miR-325/ACP5 axis played a critical role to regulate HCC progression, and targeting this newly identified signaling with oemiR-325 led to suppression of HCC progression.Taken together the data from HCC is one of the most lethal malignant tumors in the world. In 2019, it is estimated that there will be 42030 new cases of liver or intrahepatic bile duct tumors and 31780 deaths from HCC in the United States The role of androgen/AR signaling in HCC initiation and progression appears to be contradictory. Studies with genetically modified animals with altered AR expression indicated that AR promoted HBV-, HCV-, and carcinogen-induced HCC initiation in vitro and in vivo experiments, which strongly proved miR-325's negative roles in regulating tumor progression.MicroRNA is a kind of non-coding RNA with 18-25 nucleotides in length, and it has been proven to induce messenger RNA degradation or inhibit protein translation to negatively regulate the expression of target genes ACP5 can promote cell movement by regulating focal adhesion kinase phosphorylation, which plays a vital role in bone resorption and osteoclast differentiation. Scott et al. showed that ACP5 promotes the invasion and distal metastases of melanoma and breast cancer cells In conclusion, AR may decrease HCC progression via altering miR-325/ACP5 signaling, and miR-325 can target the 3'UTR of ACP5-mRNA to suppress its expression. A potential therapy to target this newly identified signaling may help improve the treatment to better suppress the HCC progression.Supplementary figure S1.Click here for additional data file.


O-benzylhydroxylamine to a benzaldehyde-tethered peptide-type hydrogelator in the presence of lipid-type nanofibers. We also succeed in construction of a parallel self-sorting network through deceleration of seed formation using a slow oxime exchange reaction. Through careful observation, the formation of peptide-type seeds and nanofibers is shown to predominantly occur on the surface of the lipid-type nanofibers via highly dynamic and thermally-fluctuated processes.Self-sorting double network hydrogels comprising orthogonal supramolecular nanofibers have attracted attention as artificially-regulated multi-component systems. Regulation of network patterns of self-sorted nanofibers is considered as a key for potential applications such as optoelectronics, but still challenging owing to a lack of useful methods to prepare and analyze the network patterns. Herein, we describe the selective construction of two distinct self-sorting network patterns, interpenetrated and parallel, by controlling the kinetics of seed formation with dynamic covalent oxime chemistry. Confocal imaging reveals the interpenetrated self-sorting network was formed upon addition of  Regulation of self-sorted nanofiber network patterns in double network hydrogels comprising supramolecular nanofibers is considered as key for potential applications. Here, the authors describe a selective construction of two distinct self-sorting network patterns, by controlling the kinetics of seed formation with dynamic covalent chemistry. However, recent evidence indicates that the interplay through physical contact among these self-sorted cytoskeletal fibers is also essential for key cellular functions including cell motility and mechano-responsiveness4. For instance, microtubule polymerization guided by a bundle of actin filaments occurred in migratory cells to regulate the turnover of stress fibers and cell motility5. Such precise control of interactions between self-sorted nanofibers  may give us important clues for the design of novel biomimetic soft materials.In living cells, cytoskeletons, such as actin filaments and microtubules, form orthogonal supramolecular nanofibers through self-sorting phenomena8. The elaborate design of hydrogelators allows for construction of stimulus-responsive hydrogels, which are promising scaffolds for drug release matrices and regenerative medicine. Self-sorting double network (SDN) hydrogels composed of orthogonal supramolecular nanofibers have recently attracted considerable attention because they provide rational integration of multiple functions38. While the control of self-sorting phenomena at the monomer level (self-sorting vs coassembly) was recently achieved37, the precise control of network patterns of self-sorted nanofibers remains challenging as it involves a higher hierarchical level of self-sorting events. In addition, evaluation of the relative spatial position of such networks is still extremely difficult. For example, several researchers reported self-sorted donor and acceptor supramolecular nanofibers containing p-n heterojunctions and conducted structural characterization by spectroscopy, TEM and/or SEM47. However, the explicit discrimination of the network patterns of the self-sorted nanofibers was impossible by these methods48. Very recently, Adams and coworkers attempted to control the network structure of self-sorted nanofibers by changing the formation kinetics and investigated the resulting network structure by small-angle neutron scattering and electron paramagnetic resonance spectroscopy47. Although valuable, these measurements gave only indirect (ensemble) structural information, which is insufficient to determine the network structure in detail. To date, there are no reliable analytical methods useful to investigate self-sorting network patterns, thus, the factors controlling the network patterns have not yet been clearly addressed.Supramolecular hydrogels consist of nanofibrous structures that are formed through self-assembly of low-molecular-weight gelatorsHerein, we describe the construction of two distinct self-sorting network patterns  by controlling the kinetics of seed formation Fig. . Dynamic64, we designed a new peptide-type hydrogelator, Ald-F(F)F, that has a benzaldehyde moiety as a reaction handle at the N-terminus of a self-assembled diphenylalanine sequence F through oxime bond formation would allow for in situ nanofiber formation and the resultant hydrogelation. To test our idea, we initially examined the gelation property of Ald-F(F)F and the more hydrophobic BnOx-F(F)F, which was synthesized by mixing Ald-F(F)F and O-benzylhydroxylamine. The slightly opaque hydrogels were formed by heating a suspension of Ald-F(F)F or BnOx-F(F)F in 100 mM MES (pH 6.0) until they dissolved, followed by cooling to room temperature F and BnOx-F(F)F were determined by the tube inversion method to be 8.6 mM and 1.3 mM, respectively, indicating that BnOx-F(F)F is a better hydrogelator than Ald-F(F)F. We next investigated in situ oxime formation-induced hydrogelation by addition of O-benzylhydroxylamine to a solution of Ald-F(F)F  F was converted to BnOx-F(F)F F hydrogel  increased from 1629 to 8554 Pa upon treatment of O-benzylhydroxylamine  Fig. . An opaqF)F Fig. . Also, t67. The fluorescent probe NP-Alexa647, which contains a diphenylalanine self-assembled motif similar to Ald-F(F)F, was used to stain the peptide-type nanofibers F and NP-Alexa647 demonstrated that the well-entangled nanofibrous structure formed upon addition of O-benzylhydroxylamine, which closely corresponded to the sol–gel transition F in the presence of lipid-type nanofibers F and Phos-MecycC5 by heating a suspension of both hydrogelators until they dissolved followed by cooling to room temperature . CLSM imaging of the viscous solution containing Ald-F(F)F, Phos-MecycC5, NP-Alexa647, and NBD-cycC6 allowed visualization of the fibrous structures of Phos-MecycC5 stained with NBD-cycC6 but not Ald-F(F)F F) stained with NP-Alexa647 newly appeared and these two fibers formed a self-sorting double network within 1 h F/NP-Alexa647 fibers, which was also supported by the low Pearson’s correlation coefficient (0.14) F was converted to BnOx-F(F)F whereas Phos-MecycC5 remained intact, suggesting that Phos-MecycC5 showed negligible effects on the oxime formation and the subsequent generation of BnOx-F(F)F nanofibers F Fig. , top. Up1 h Fig. , bottom.14) Fig. 70. 3D stBnOx-F(F)F and Phos-MecycC5, we examined the self-sorting behavior of BnOx-F(F)F and Phos-MecycC5 by macroscopic observation and rheological analysis. We prepared a mixture of BnOx-F(F)F and Phos-MecycC5 by heating until dissolving followed by cooling to room temperature (termed heat-cool protocol). In this case, a suspension containing a white precipitate was formed instead of a hydrogel F and Phos-MecycC5 F gel , suggesting that integration of the peptide- and lipid-type networks increased the mechanical property of the hydrogel. Furthermore, CLSM imaging revealed that the suspension consisted of a complex mixture containing spherical aggregates stained with NBD-cycC6, peptide-type nanofibers stained with NP-Alexa647, and nanofibrous structures stained with both NP-Alexa647 and NBD-cycC6 F and Phos-MecycC5 that were prepared separately F using the in situ oxime formation is crucial for construction of the interpenetrated SDN.To support the importance of in situ peptide fiber formation (termed oxime-formation protocol) by dynamic covalent chemistry for the interpenetrated SDN of cC6 Fig. . A suspeO-benzylhydroxylamine to the solution containing Ald-F(F)F, Phos-MecycC5, NP-Alexa647, and NBD-cycC6 on a glass bottom dish, and then started CLSM imaging. To our surprise, careful real-time observation revealed two distinct processes in the formation of peptide-type nanofibers F generation may enable preferential nucleation/elongation proximal to the lipid-type nanofibers leading to construction of a parallel self-sorting network. To suppress the rate of the nucleation process, we sought to use a two-step oxime exchange reaction. As shown in Fig. Ald-F(F)F hydrogel to produce an oxime peptide, CaOx-F(F)F. Since CaOx-F(F)F is more hydrophilic at its N-terminus, it should exhibit poor hydrogelation compared with Ald-F(F)F according to our previously established design principle65. Indeed, the critical gelation concentration of CaOx-F(F)F was determined to be 26 mM, which is much higher than that of Ald-F(F)F (8.6 mM) F via an oxime exchange reaction (hereafter we refer to this experiment as the oxime-exchange protocol). We conducted in situ imaging of fiber degradation and formation by the two-step oxime exchange process F/Phos-MecycC5 hydrogel stained with NP-Alexa647 and NBD-cycC6 revealed that Ald-F(F)F and Phos-MecycC5 nanofibers formed the interpenetrated SDN structure F and Phos-MecycC5 are a good self-sorting pair. Upon addition of carboxymethoxylamine (1.2 eq) to this self-sorting network, the peptide-type nanofibers completely disappeared in 4 h, whereas Phos-MecycC5 nanofibers were almost unchanged  and their localization appeared to overlap with the Phos-MecycC5 nanofibers F/Phos-MecycC5 and of Ald-F(F)F/Phos-MecycC5  proceeded in 98 and 30% yield, respectively, whereas Phos-MecycC5 remained unchanged F to BnOx-F(F)F in the second step (4.9 mM) was higher than the CGC of BnOx-F(F)F (1.3 mM), and the resultant concentration of BnOx-F(F)F was comparable with that in the oxime-formation protocol (4.3 mM). As controls, buffer treatment at each step induced neither collapse nor formation of the peptide-type nanofibers  and the second oxime exchange (with Ald-F(F)F, Phos-MecycC5, and O-benzylhydroxylamine F was essential for formation of the parallel SDN; a suspension with white precipitate was formed by the direct mixing of CaOx-F(F)F and Phos-MecycC5 with the heat-cool protocol F and Phos-MecycC5. In this case, however, the interpenetrated SDN was mainly formed F and Phos-MecycC5 showed the mixed network structure of interpenetrated and parallel SDNs F and Phos-MecycC5 with O-benzylhydroxylamine never induced transformation to the parallel SDN F to CaOx-F(F)F did not proceed upon addition of an excess amount of carboxymethoxylamine, implying that the self-assembled structure stabilizes BnOx-F(F)F probably due to its tight packing and/or the hydrophobic microenvironment F, therefore the nucleation proceeded in both sites. In addition, the slower kinetics of BnOx-F(F)F generation by the oxime exchange allowed for preferential nucleation on the energetically-favorable surface of the Phos-MecycC5 nanofibers. These behaviors seem to be consistent with the physics of nucleation barriers as recently demonstrated by liquid droplet formation in live cells with optogenetics73. The present example demonstrates that controlling the nucleation process would be a promising strategy to construct an artificially regulated network pattern of supramolecular hydrogels.On the basis of the real-time CLSM observations, it was clear that there were critical differences in the kinetics and spatial locations of the peptide-type nanofiber formation between the oxime-formation and oxime-exchange protocols. In the oxime-formation protocol, the short peptide-type nanofibers appeared 1.5 min after addition of BnOx-F(F)F/Phos-MecycC5 (prepared by the oxime-formation protocol) and Ald-F(F)F/Phos-MecycC5 did not show any significant changes at least for 3 days F or BnOx-F(F)F powder in 100 mM MES, pH 6.0 was heated by a heating gun  until dissolving. The resultant hot solution was cooled to room temperature (rt) and incubated for 24 h. The state (gel or sol) of the sample was judged by the tube inversion test. The assay conditions were referred in the figure captions.A suspension of an Ald-F(F)F powder (4.3 mM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. The resultant hot solution was cooled to rt and incubated for 1 h. To this resultant solution, a solution of O-benzylhydroxylamine  was added and incubated for 1 h. The state (gel or sol) of the sample was judged by the tube inversion test. The assay conditions were referred in the figure captions.A suspension of an Ald-F(F)F powder (17.3 mM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. The resultant hot solution was cooled to rt and incubated for 1 h. To this resultant hydrogel, a 10× solution of O-benzylhydroxylamine  was added and incubated for 1 h. The hydrogel was analyzed by rheological and HPLC analyses.A suspension of an Ald-F(F)F (4.3 mM) and NP-Alexa647 (4.0 µM) with/without Phos-MecycC5 (2.4 mM) and NBD-cycC6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. After cooling to rt, the resultant mixture (18 µL) was transferred to a glass bottom dish (Matsunami) and incubate at rt for 1 h in the presence of water to avoid dryness. To the resultant solution, a solution of O-benzylhydroxylamine  or buffer was added. After incubation for 1 h, CLSM imaging was conducted.The suspension of BnOx-F(F)F (4.3 mM) and Phos-MecycC5 (2.4 mM) with/without NP-Alexa647 (4.0 µM) and NBD-cycC6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. The resultant mixture was cooled to rt and incubated at rt for 24 h. The state (gel or sol) of the sample was judged by the tube inversion test. The obtained suspension (containing fluorescent probes) was moved to a glass bottom dish and observed by CLSM imaging.The suspension of BnOx-F(F)F/NP-Alexa647 (8.6 mM and 8.0 µM) and Phos-MecycC5/NBD-cycC6 (4.8 mM and 8.0 µM) were separately heated by a heating gun until dissolving. The equal volume of the resultant hot solutions were immediately mixed and incubated at rt for 1 h. The state (gel or sol) of the sample was judged by the tube inversion test. The obtained suspension (containing fluorescent probes) was moved to a glass bottom dish and observed by CLSM imaging.The suspensions of Ald-F(F)F (17.3 mM) and NP-Alexa647 (4.0 µM) with Phos-MecycC5 (2.4 mM) and NBD-cycC6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. After cooling to rt, the resultant mixture (10 µL) was transferred to a glass bottom dish (Matsunami) before gelation. After incubation at rt for 1 h in the presence of water to avoid dryness, CLSM imaging was conducted. To the resultant hydrogel, a solution of carboxymethoxylamine  or buffer was added. After incubation for 4 h, CLSM imaging was conducted. Subsequently, O-benzylhydroxylamine  or buffer was added to the resulting solution. After incubation at rt for 48 h, CLSM imaging was conducted.The suspension of CaOx-F(F)F (17.3 mM) and Phos-MecycC5 (2.4 mM) with/without NP-Alexa647 (4.0 µM) and NBD-cycC6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. The resultant mixture was cooled to rt and incubated at rt for 1 h. The state (gel or sol) of the sample was judged by the tube inversion test. The obtained suspension (containing fluorescent probes) was moved to a glass bottom dish and observed by CLSM imaging.The suspension of Ald-F(F)F (4.3 mM) with/without NP-Alexa647 (4.0 µM) were added a 10× solution of O-benzylhydroxylamine (43 mM). The resultant mixture was incubated at 30 °C for 1 h. A 10× solution of carboxymethoxylamine (430 mM) was added to the hydrogel and incubated at 30 °C for 24 h. The resulting samples were analyzed by HPLC analysis and CLSM imaging. The state (gel or sol) of the sample was judged by the tube inversion test.To a solution of BnOx-F(F)F (4.3 mM) and Phos-MecycC5 (2.4 mM) with/without NP-Alexa647 (4.0 µM) and NBD-cycC6 (4.0 µM) were heated by a heating gun until dissolving. The resultant hot solutions were incubated on a heating plate at 100 °C for 5 min, then the setting temperature was changed from 100 °C to 30 °C, and the sample was incubated for 30 min on the heating plate. The state (gel or sol) of the sample was judged by the tube inversion test. The sample was analyzed by CLSM imaging.The suspension of 3CN containing 0.1% TFA, B: H2O containing 0.1% TFA).The preparation of the hydrogels and solutions was described above. The samples (133 µL) were dissolved by cold DMF (800 μL) and a DMSO solution of fluorescein . The resultant mixture was filtered with membrane filter (diameter: 0.45 μm), and then analyzed by RP-HPLC  were carefully taken out from the PDMS mold and put onto the stage of a rheometer  with a parallel plate geometry. Strain sweep data were obtained using shear mode at a frequency of 10 rad/s, and linear dynamic viscoelasticity were measured in shear mode at 0.3 or 1.0% strain amplitude for frequency sweep.The elongation distance of the peptide-type nanofiber was estimated by comparing the successive two images of the time-lapse imaging with Fiji. The velocity was calculated by dividing the elongation distance by interval of time-lapse imaging. In total, 50 and 120 elongated nanofibers were randomly selected (Supplementary Figs. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


In this study, we investigated the effects of co-inoculation with a diazotrophic bacterium (Paenibacillus beijingensis BJ-18) and a P-solubilizing bacterium (Paenibacillus sp. B1) on wheat growth, plant and soil total N, plant total P, soil available P, soil nitrogenase activity and the relative expression of nifH in plant tissues. Co-inoculation significantly increased plant biomass  and plant N content  and P content . Co-inoculation also significantly increased soil total N (12%), available P (9%) and nitrogenase activity (69%) compared to P. beijingensis BJ-18 inoculation alone. Quantitative real-time PCR analysis showed co-inoculation doubled expression of nifH genes in shoots and roots. Soil nitrogenase activity and nifH expression within plant tissues correlated with P content of soil and plant tissues, which suggests solubilization of P by Paenibacillus sp. B1 increased N fixation in soils and the endosphere. In conclusion, P solubilizing bacteria generally improved soil available P and plant P uptake, and considerably stimulated BNF in the rhizosphere and endosphere of wheat seedlings.Phosphate (P) availability often limits biological nitrogen fixation (BNF) by diazotrophic bacteria. In soil, only 0.1% of the total P is available for plant uptake. P solubilizing bacteria can convert insoluble P to plant-available soluble P (ionic P and low molecular-weight organic P). However, limited information is available about the effects of synergistic application of diazotrophic bacteria and P solubilizing bacteria on the nitrogenase activity of rhizosphere and  In this ammonia , and fix ammonia . In low  ammonia , 2011 an ammonia .Although there are large amounts of organic and inorganic P in soils, only 0.1% total P is available for plants , resultiPaenibacillus strains have potential as biofertilizers due to their capacity to form endospores, which can survive for long time periods under adverse conditions (P. beijngensis BJ-18 from the rhizosphere of wheat. This plant growth promoting rhizobacteria (PGPR) exhibits high nitrogenase activity  and inorganic P and promote the growth and improve nutrition of maize and tomato. Obervations of GFP-labbled Paenibacillus sp. B1 shows that this P-solubilizing strain effectively colonizes maize root surface and in epidermal and cortical tissues  pot experiment with different inoculation treatments was conducted. We investigated the effects of experimental treatments on (1) host plant biomass, (2) rhizosphere soil total N, available P and nitrogenase activity, (3) plant tissue total N, total P and relative expression of nifH and (4) the Pearson’s correlation coefficient between the nitrogenase activity/nifH expression and P nutrition of the soil/plant tissues.To test the hypothesis that these two PGPR, with complementary traits, show synergy, a factorial wheat (P. beijingensis BJ-18 (accession number: JN873136) was isolated from rhizosphere soil of wheat (Paenibacillus sp. B1 (accession number: KY111475), isolated from the maize rhizosphere, is a novel species with P solubilizing ability and produce IAA  containing 100 mL of Luria Bertani broth and cultured at 30 °C overnight. After growth, the bacterial cells were harvested by centrifugation at 4,000×g for 5 min and diluted with deionized water to 108 cells/mL.of wheat . Paenibaduce IAA . P. beijPaenibacillus sp. B1 (B1) (2) wheat seedling inoculated with P. beijingensis BJ-18 (BJ-18) (3) wheat seedling inoculated with Paenibacillus sp. B1 and P. beijingensis BJ-18  (4) non-inoculated wheat seedlings as a negative control (CK) and (5) wheat seedlings inoculated with P. beijingensis BJ-18 and watered with nutrient solution containing soluble P (BJ-18+P) as a positive control. Plants were irrigated with N-free nutrient solution  in the presence or absence of KH2PO4 where applicable (43 mL per pot).Seedling growth assays were performed in plastic pots  filled with 1 kg low nutrition-content soil. The collection and physicochemical properties of soil were described by 8 cells/mL) for 30 min to facilitate colonization. For CK treatment, the plant seedlings were soaked in sterilized deionized water. Then the inoculated and non-inoculated seedlings were respectively transplanted into pots (3 hills per pot and 15 seedlings per hill). Sixteen millilitres of the bacteria suspension were applied to pots containing inoculated seedlings and 16 mL of sterile water was applied to pots containing non-inoculated seedlings on day 7. All treatments had five replicates. Pots were placed in the greenhouse under optimum conditions . The seedlings were watered every 5 days.Plump seeds of wheat “Jimai 22”  were soaked in 10% sodium hypochlorite for 10 min, and rinsed thrice with sterilized water. The sterilized wheat seeds were placed on the sterile plates containing moist filter papers and cultivated for 3–5 days at room temperature (25 °C). After germination, vigorous and homogeneous wheat seedlings were chosen. For inoculation, the wheat seedlings were soaked in bacteria suspension .2SO4–H2O2 mixture at 370 °C and then total N was determined using the modified Kjeldahl method . The RNA concentration was measured spectrophotometrically at 220 nm using a spectrophotometer . RNA was digested with DNase I , and reversely transcribed into cDNA with using PrimeScript™ RT reagent kit . qRT-PCR was conducted in an Applied Biosystems 7500 Real-Time System  using the SYBR® Premix Ex TaqTM . The nifH primers were chosen for qRT-PCR  was conducted using SPSS 20.0 . The Pearson’s correlation coefficient was analyzed using GraphPad Prism 7.00 . Graphs were prepared using GraphPad Prism 7.00 and SigmaPlot software version 12.5 .Compared to CK treatment, B1, BJ-18, Mix and BJ-18+P treatments significantly increased wheat root length  and 1D iTotal N of wheat roots in BJ-18, Mix and BJ-18+P were significantly increased by 35%, 71% and 36%, respectively, compared to CK treatment . CompareCompared to CK treatment, BJ-18, Mix and BJ-18+P treatments significantly increased SPAD value. Mix and BJ-18 treatments were not significantly different from each other in chlorophyll content .Compared to CK treatment, treatments of BJ-18, Mix and BJ-18+P significantly increased total N of rhizosphere soil by 20%, 34% and 33%, respectively . B1, MixnifH were significantly up-regulated in wheat roots of BJ-18, Mix and BJ-18+P treatments by 18, 63 and 54 fold, respectively, and in wheat shoots of BJ-18, Mix and BJ-18+P treatments by 9, 31 and 17 fold, respectively, compared to those in the CK treatment  and the relative expression of 10.7717/peerj.9062/supp-1Supplemental Information 1Click here for additional data file.


Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal β-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus. Staphylococcus aureus (S. aureus) is a serious burden to public health as it is a leading cause of antibiotic-resistant infections worldwide. Vaccines aimed at targeting S. aureus have failed in clinical trials, and the reason for this lack of success remains unclear. As this pathogen continues to rapidly spread on a global scale, it is vital that new approaches to S. aureus vaccination are developed. In this study, we show that activation of antifungal immunity with a β-glucan particle vaccine can be harnessed to direct protection against S. aureus systemic infections in mice. This work broadens the use of the β-glucan particle vaccine system for potential use as a much-needed vaccine targeting S. aureus. Staphylococcus aureus is both a human commensal and a formidable opportunistic pathogen. As a commensal, S. aureus can be found in places such as the anterior nares, throat, skin, and gastrointestinal tract [S. aureus in the nasal mucosa [S. aureus can cause a range of diseases including skin and soft tissue infections (SSTIs), endocarditis, sepsis, pneumonia, osteomyelitis, bacteremia, and abscesses in organ tissues [S. aureus bacteremia mortality remains at a high rate of 15–50%, despite the use of novel antibiotics and emphasis on disease surveillance and prevention [S. aureus are becoming increasingly prevalent, with the most common, methicillin-resistant S. aureus (MRSA), emerging in hospitals and communities around the world. These strains infect healthy individuals and immunocompromised patients alike [al tract –3. It isl mucosa , 4. As a tissues . Moreoveevention . AntibioS. aureus does not usually promote robust protective immune responses, and individuals are not typically protected from subsequent S. aureus infections [S. aureus is an urgent and crucial public health need. Unfortunately, vaccines tested in clinical trials thus far have proved unsuccessful in providing protection [S. aureus infections [Unlike many bacterial infections, fections . Hence, otection –11. Thesfections , 13, sugfections , 14.S. aureus infections [S. aureus infections [S. aureus [S. aureus also predispose individuals to fungal infections. Patients with HIV are more susceptible to fungal infections, especially chronic mucocutaneous candidiasis (CMC) [S. aureus infections.Individuals that have defects in CMI such as HIV, defective IFNγ production, or treatment with high levels of glucocorticoids experience more fections . Patientfections , 16. Due. aureus . Intriguis (CMC) . FurtherS. aureus infections [S. aureus infection. Antifungal immunity might be harnessed to activate such responses.Th1 and Th17 subsets have been shown to be especially vital for antifungal responses in animal models and in humans –20. Th1 fections . FailureSaccharomyces cerevisiae can act as an adjuvant by stimulating Dectin-1 and CR3. The hollow structure of GPs allows them to be loaded with payloads such as proteins or peptide antigens, DNA, siRNA, nanoparticles, or other small molecules for diverse purposes, including vaccine development [+ T cells to Th1 and Th17 subsets [Innate pattern recognition receptors (PRRs), which recognize pathogen associated molecular patterns (PAMPs), are responsible for initial sensing of pathogens and tailor inflammatory immune responses to direct ensuing adaptive responses . One sucelopment . Antigen subsets , 31–37.S. aureus infections, we hypothesized that a glucan particle vaccine could provide protective immunity to S. aureus by activating antifungal-tailored immunity though the β-glucan particle while delivering S. aureus antigens. Here we demonstrate that immunization of mice with a GP + S. aureus antigen vaccine protects mice against systemic infection with S. aureus. Multiple immune cell types including dendritic cells phagocytose the vaccine in vivo in the peritoneal cavity, and these professional antigen presenting cells are vital for stimulating strong adaptive T cell responses [+ T cells to the Th1 and Th17 subsets and the production of antigen-specific antibodies, consistent with previous studies using ovalbumin-loaded GPs [+ T cells are critical for vaccine-induced protection. Finally, the GP + S. aureus antigen vaccine elicited long-term protection as mice had reduced bacterial burden in the spleen and kidneys and detectable antibody levels eight weeks after immunization.Given the connection between patients with specific immune defects and their increased risk for fungal and esponses . Moreoveaded GPs , 29. WhiS. aureus vaccine is still a matter of debate [S. aureus virulence factors and survival mechanisms that may be effective at stopping the bacteria at different stages and sites of infection [S. aureus strains and identified antigens promoting the best protection [S. aureus vaccination studies providing protection from lethal i.v. challenge, abscess formation, arthritis, and necrotic wound infection . Add. AddS. aotection , 47. As S. aureus vaccine in mice  as previously created and described by the Levitz and Ostroff groups [+ T cells  in the context of a f groups , 26, 29.f groups , 48–50.  T cells . Mouse b the GPs . These r the GPs . Moreove the GPs . These rnfection .+ at 6 hours and over 70% at 24 hours post-stimulation. 4X-SA-GP also induced maturation of BMDCs as seen by upregulation of surface MHC-II and CD86  dose of 5x107 FITC-4X-SA-GP or unlabeled 4X-SA-GP as a control. We collected cells by peritoneal lavages at 24, 48, and 72-hours post-injection and analyzed FITC+ immune cells by flow cytometry  followed by a four-week resting period (S. aureus (USA300) by i.p. injection of 2x107 colony forming units (CFUs), and we measured bacterial burdens in the spleen and kidney after 24 hours. Mice vaccinated with 4X-SA-GP had significantly reduced bacterial burden in the spleen compared to mice vaccinated with the vehicle control . We collected supernatants after three days of culture for analysis by ELISA, and we restimulated the cells with PMA and ionomycin for analysis by intracellular cytokine staining for IFNγ and IL-17. Flow cytometry revealed a significant increase in the frequency of IL-17+ CD4+ T cells from the spleen of 4X-SA-GP vaccinated mice when stimulated with the protein mixture and with HK-SA in comparison to the CD4+ T cells of mice vaccinated with empty-GPs or PBS control . By comparison, single dose vaccination with 4X-SA-GP also induced antigen-specific CD4+ T cell responses, though the Th17 responses were not as strong  was used. Mice were immunized three times as above, and an additional cohort of mice was immunized by the same route and schedule with matching amounts of the four approach . Taken tS. aureus vaccine should most likely elicit both antibody and CD4+ T cell responses [S. aureus and assessed spleen and kidney CFUs after 24 hours. There were no differences in the bacterial burdens in the spleen  received an i.p. injection of either anti-CD4+ antibody or the corresponding isotype control antibody four weeks post-vaccination. To ensure depletion of the CD4+ T cells, we measured CD4+ T cell percentages prior to infection in peripheral blood mononuclear cells (PBMCs) isolated from each group of mice and post-infection in the MLNs by flow cytometry , have rapidly emerged worldwide, infecting immunocompromised and healthy individuals alike, making it a major public health threat [S. aureus have failed in clinical trials [S. aureus vaccine emerge. The observation that conditions that predispose a person to S. aureus infections often also predispose to fungal infections suggests that the elements of immunity that are effective against S. aureus share much in common with the elements of immunity that promote defense against fungal infections. We therefore hypothesized that a vaccine based on activating antifungal innate immune responses in the context of delivery of S. aureus antigens might be productive. In this study, we developed and tested in mice a vaccine candidate consisting of fungal β-glucan particles loaded with four S. aureus protein antigens (4X-SA-GP).h threat . Vaccinel trials  and the S. aureus protein antigens we used, ClfA and MntC have and are being used (together with capsular polysaccharides) in vaccines that have gone on to clinical trials. In this case, the vaccine employs a non-toxic mutant of diphtheria toxin cross-reacting material 197 (CRM197) as an adjuvant, but it has so far not proven effective at preventing infection. The vaccine generates antibodies to ClfA and MntC that would appear to be effective in vitro [Of the four in vitro . A variein vitro . A workiin vitro .S. aureus. The vaccine also induced strong IgG1 and IgG2c antibody responses to the S. aureus proteins, although it was not clear that these antibody responses were sufficient to mediate protection from infection.Vaccinating mice with 3 doses of 4X-SA-GP induced long lasting (at least 8 weeks) cellular and humoral antigen specific immune responses. Antigen-specific T cell responses were polarized towards Th1 and Th17 as would be anticipated for the adjuvant activity of β-glucan, and these T cell responses were essential for vaccine-induced protection from systemic infection with S. aureus infection that was induced by β-glucan particles having no S. aureus antigens. This immunity wore off after 8 weeks, but the antigen specific immunity induced by antigen-loaded particles persisted. It is therefore possible that the β-glucan particles alone elicit some short-lived non-antigen-specific inflammatory immunity by which innate immune cells show enhanced responsiveness. Future studies could be designed to characterize the cellular and molecular signatures associated with this temporary protection, but the antigen-specific responses are longer-lived.Four weeks after completing the vaccination, animals retained some variable non-specific enhanced resistance to Candida albicans vaccine discovered that their vaccine provided cross-protection from S. aureus [Candida agglutinin-like sequence 3 (Als3) adhesin/invasin as the antigen and is mixed with alum as an adjuvant. While the vaccine induces good antibody production against Als3 that cross react with S. aureus cell walls, protection in mouse models of infection required T cell mediated immunity , not B cell immunity [+ T cells to both the Th1 and Th17 subsets in response to the 4X-SA-GP may make this approach more desirable compared to vaccines that polarize T cells to only one main subset. Th17 cells appear to play a major role in eliciting protection for skin, respiratory, and mucocutaneous infections [+ T cells to both these subsets by 4X-SA-GP immunization may help ensure that an effective T cell-mediated downstream response will occur upon infection. Previous studies have indicated that S. aureus-specific T cells that stimulate phagocyte recruitment and activation through the production of the proinflammatory cytokines IFNγ and/or IL-17 are important for immunity to S. aureus [Some years ago, a group of investigators working on development of a . aureus . The vacimmunity , 63. Polfections , 65, whifections . Therefo. aureus , 66.Cryptococcus-derived alkaline extracts protected animals from a lethal challenge with Cryptococcus neoformans and Cryptococcus gattii [Aspergillus fumigatus infection [S. aureus, and they demonstrate the potential GPs have as a vaccine to induce protective cellular immunity. The similarities between the immune responses needed for antifungal and anti-staphylococcal immunity suggest that similar vaccination strategies might be effective, and further studies will be required to more completely optimize a GP-based S. aureus vaccine.The GP vaccine platform has been previously investigated primarily in the context of developing fungal vaccines , 67 due s gattii . Glucan This study was performed under strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. This protocol was approved by the institutional animal use and care committee of the Cedars-Sinai Medical Center (IACUC 7174 and 8836).S. aureus infection experiments, the dominant CA-MRSA strain in the United States, USA300, specifically the Los Angeles County clone (LAC) was used [S. aureus LAC was routinely cultured in Tryptic Soy Broth. Mid-log phase bacteria sub-cultured from overnight cultures were used for experiments. Bacteria were washed in PBS twice prior to use and inocula were confirmed by CFU determination on blood agar plates. Heat-killed S. aureus was prepared by incubating a washed overnight culture of S. aureus for 45 minutes at 90°C. The bacteria were then washed to remove secreted proteins. The sterility of killed bacteria was confirmed by enumeration of CFU on an agar plate.For all was used . S. aurein vitro and in vivo experiments. Due to sex-related differences in susceptibility to S. aureus [C57BL/6 mice were purchased from Jackson Laboratories and Rag2/OT-II transgenic mice were purchased from Taconic. OT-II TCR transgenic mice and Dectin-1 knockout mice were bred and housed under specific pathogen-free conditions in the Cedars-Sinai Medical Center animal facility. Aged-matched female mice aged 8–12 weeks were used for . aureus , only fein vitro stimulation assays. CD4+ T cells were isolated from pooled spleens and lymph nodes of 8-12-week-old female OT-II or Rag2/OT-II mice using an EasySep mouse naïve CD4+ T cell Isolation kit (STEMCELL Technologies).Bone marrow-derived dendritic cells (BMDCs) from C57BL/6 and Dectin-1 knockout mice were grown as previously described  in RPMI 5 cells/well in 24-well TC plates  or 2x105 cells/well in 96 well round-bottom TC plates (for T cell co-culture). The following day after re-plating, BMDCs were stimulated for 24 hours with GP-OVA (10 particles/cell), 4X-SA-GP (10 particles/cell), or 100 ng/mL LPS (Invivogen). For cytokine analysis of BMDCs, supernatants were harvested 24 hours after stimulation, and for analysis of maturation markers, BMDCs were lifted after overnight stimulation with PBS containing 2mM EDTA, followed by immunofluorescent staining and flow cytometry.BMDCs were plated at 5x102SO4 and the plates were read at 450 nm.Enzyme linked immunosorbent assays (ELISA) were performed according to the manufacturer’s instructions for IL-6, TNFα, IL-17A, and IFNγ (BioLegend). For antibody analysis in the serum of mice, ELISA plates (Corning Costar) were coated with 5 μg per well of rClfA, rIsdA, rMntC, rSdrE at 4°C overnight. The plates were blocked with ELISA assay diluent (BioLegend) followed by incubation with the sera from vaccinated mice at dilutions of 1:1000, 1:10,000, and 1:100,000 in the assay diluent. The plates were then incubated with biotin-conjugated anti-mouse IgG1 (BioLegend) followed by streptavidin-conjugated anti-mouse IgG-HRP (BioLegend). For IgG2c, sera were incubated with anti-mouse IgG2c-HRP (SouthernBiotec). The plates were then incubated with TMB substrate solution (BD OptEIA) and the reactions were stopped with 2N HFluorophore-conjugated anti-mouse antibodies directed against the following molecules were used to stain cells: CD11b (M1/70), CD11c (N418), Ly6C (HK1.4), Ly6G (1A8), F4/80 (BM8), CD19 (1D3/CD19), I-A/I-E , CD86 (GL-1), IL-17A (TC11-8H10.1), IFNγ (XMG1.2), CD3 (145-2C11), TCRβ (H57-597), CD4 (GK1.5), TCR Vβ5.1/5.2 (MR9-4), TCR Vα2 (B20.1) . All samples were prestained with anti-CD16/CD32 (eBioscience) to block FC receptors, and Zombie fixable viability dye (BioLegend) to identify dead cells. For intracellular cytokine staining, cells were stimulated for 4 hours with Cell Activation Cocktail (BioLegend) in the presence of GolgiStop (BD Biosciences) for the last 3 hours of stimulation. Following the addition of Fc-block, viability dye and surface staining, intracellular cytokine staining was performed using the cytofix/cytoperm staining kit (BD Biosciences) according to the manufacturer’s instructions. Samples were acquired with a LSRII (BD Biosciences) and data were analyzed with FlowJo software (Tree star).6 particles were loaded onto a gradient gel and subjected to gel electrophoresis using the Novex NuPAGE Gel electrophoresis system (Invitrogen), and proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). GP-OVA blots were probed with an anti-ovalbumin antibody , followed by an HRP-conjugated secondary antibody (Jackson ImmunoResearch). Blots were incubated with ECL substrate (Pierce) and then exposed to X-ray film. 4X-SA-GP blots were probed with an anti-6xHN antibody (Clontech Laboratories) to detect the polyhistidine tag on the N-terminus of each of the recombinant proteins followed by infrared dye-conjugated secondary antibodies (LI-COR). Bands were visualized on an Odyssey imaging system (LI-COR).For assessing ovalbumin or the recombinant proteins in the glucan particles, 2x105 cells/well and were stimulated overnight with GP-No OVA or GP-OVA (10 particles/cell). The following day, the BMDCs were washed thoroughly and CFSE-labeled [+ T cells were added at a 1:5 ratio (2x105 DC: 1x106 T cells) and cells were cultured with RPMI supplemented with 10% FBS, 1 mM sodium pyruvate and 55 μM 2-mercaptoethanol. Following 4–5 days of co-culture, supernatants were harvested for cytokine analysis by ELISA and T cells were restimulated with Cell Activation Cocktail (BioLegend) and Golgistop (BD Biosciences) and stained intracellularly with fluorophore-conjugated antibodies against IL-17A and IFNγ.BMDCs were plated in 96 well round-bottom TC plates at 2x10-labeled  naïve OTSaccharomyces cerevisiae zymosan A (Sigma) with a hot-alkali treatment to remove TLR agonists as previously described [S. aureus proteins (with or without TRITC or FITC-labeling) as previously described [in vitro or in vivo use. Empty-GPs were prepared the same as the loaded-GPs minus the addition of any protein antigen.β-glucan particles were prepared by treating escribed . Brieflyescribed , 51. GluCoding sequences for ClfA, IsdA, MntC, and SdrE were PCR amplified without the signal peptide or sorting signal  was determined via CountBright Absolute Counting Beads (Invitrogen).Mice were injected intraperitoneally with 5x106 cells/well. For specific stimulation, cells were cultured with a mixture of the 4 recombinant proteins (10 μg/protein per mL) or heat-killed S. aureus at an MOI of 5. Supernatants were collected after 72 hours for analysis of cytokines by ELISA. For intracellular cytokine analysis, the cells were stimulated with Cell Activation Cocktail (BioLegend) for 4 hours and Golgistop (BD Biosciences) for the last 3 hours of stimulation. Intracellular cytokines (IL-17A and IFNγ) were then analyzed by flow cytometry.Spleens were isolated 5 days after the final vaccination of mice with PBS, empty-GP, or 4X-SA-GP. Red blood cells were lysed and cells were resuspended in RPMI supplemented with 10% FBS, 1 mM sodium pyruvate, and 55 μM 2-mercaptoethanol and plated at 3x107 GPs). Mice were rested for 5 days before the spleen and MLNs were harvested for the T cell restimulation assay. For the infection model, mice were rested for 4 weeks after the one vaccination and were then inoculated with 2x107 CFU of S. aureus (LAC USA300) via i.p. injection. Mice were euthanized 24 hours after the infection and spleen and kidneys were harvested, homogenized, and plated on blood agar plates. CFUs were enumerated after overnight incubation at 37°C.Single vaccination model: Mice were vaccinated once with PBS as a control, empty-GPs, or 4X-SA-GP (5x107 GPs/dose). For comparison to alum, mice were immunized with payload-matched amounts of S. aureus antigens (50 μg/injection) mixed with alum (1 mg/injection). The quantities of the glucan particle-loaded recombinant proteins were estimated by knowing the amount of protein added to a particle preparation and subtracting the amount left over in supernatants. Mice were rested for 5 days before the spleen and MLNs were harvested for the T cell restimulation assay. For the infection model, mice were rested for 4 or 8 weeks (where indicated) after the final vaccination and were then inoculated with 2x107 CFU of S. aureus (LAC USA300). Mice were euthanized 24 hours after the infection and spleen and kidneys were harvested, homogenized, and plated on blood agar plates. CFUs were enumerated after overnight incubation at 37°C.Triple vaccination model: Mice were vaccinated once a week for 3 weeks with PBS as a control, empty-GPs, or 4X-SA-GP (5x107 GPs/dose). Two weeks after the final vaccination, serum was collected from all of the mice and pooled according to the vaccination group. Recipient mice received an i.p. injection of 150 μL of the pooled serum on day -1 and were then inoculated with 2x107 CFU of S. aureus (LAC USA300) via i.p. injection the following morning on day 0. Mice were euthanized 24 hours after the infection and spleen and kidneys were harvested, homogenized, and plated on blood agar plates. CFUs were enumerated after overnight incubation at 37°C.Mice were vaccinated once a week for 3 weeks with PBS as a control, empty-GPs, or 4X-SA-GP (5x107 GPs/dose). Mice were rested for 4 weeks and then received an i.p. injection with an anti-CD4+ antibody  300 μg in 500 μl PBS on day -1 and 100 μg in 500 μl PBS on day 0 as previously described [7 CFU of S. aureus (LAC USA300) via i.p. injection. Mice were euthanized 24 hours after the infection and spleen and kidneys were harvested, homogenized, and plated on blood agar plates. CFUs were enumerated after overnight incubation at 37°C. PBMCs were isolated from the blood of mice and pooled from all groups of mice on day 0 prior to infection and were stained with the surface markers CD3, CD4, and TCRβ and analyzed by flow cytometry to assess the efficacy of the CD4+ T cell depletion. MLNs were harvested on day 1, after the infection, and the isolated cells were stained for the surface markers CD3, CD4, and TCRβ and analyzed via flow cytometry to assess that the CD4+ T cells were still depleted post-infection.Mice were vaccinated once a week for 3 weeks with PBS as a control, empty-GPs, or 4X-SA-GP , or Mann-Whitney U test using GraphPad Prism software. All statistical details of experiments such as the number of replicates can be found in the figure legends. Where present, error bars indicate the mean +SD. p values less than 0.05 are considered significant.S1 Fig7 4X-SA-GPs were loaded into the lane.(A) Schematic for the generation of 4X-SA-GP. Glucan particles were loaded with the purified, his-tagged, recombinant proteins: rClfA, rIsdA, rMntC, and rSdrE. (B) Coomassie stained SDS-PAGE gel of the 4 purified recombinant proteins: Lane 1, rClfA (98.3 kDa), Lane 2, rMntC (36.2 kDa), Lane 3, rIsdA (34.1 kDa), Lane 4, rSdrE (118.6 kDa). The approximate molecular weight of each protein is shown in parentheses. All lanes contain approximately 10 μg of protein. (C). Western blot analysis of 4X-SA-GP using an anti-6xHN antibody to probe for the his-tagged proteins within the GPs. 2x10(TIF)Click here for additional data file.S2 Fig+ T cells under non-polarizing conditions. (A) Representative flow cytometry plots show the degree of Th1 (IFNγ+) and Th17 (IL-17+) T cell differentiation under each condition of BMDC activation. (B) Representative flow cytometry histograms demonstrate the degree of T cell polarization corresponding to the data above with the pink histogram from the co-culture with no stimulation used as a reference point for the percentage of proliferation. (C-D) Bar graphs quantifying the percentage of IL-17+ (C) and IFNγ+ (D) OT-II cells and the percentage of CFSE proliferation (E). (F-G) Supernatants from the co-culture were collected on day 5 and analyzed for production of IL-17 (F) and IFNγ (G) by ELISA. Flow cytometry percentages and ELISA data and their standard deviation are representative of two replicates/condition. Data are representative of two experiments.(A-G) BMDCs stimulated with GP-No OVA or GP-OVA (loaded with various concentrations of OVA) were used to activate naïve CFSE-labeled OT-II CD4(TIF)Click here for additional data file.S3 FigS. aureus purified recombinant proteins (proteins) and heat-killed S. aureus (HK-SA) for 3 days. . After 3 days, the splenocytes were stimulated with phorbol myristate acetate (PMA) and ionomycin and then analyzed by flow cytometry after intracellular cytokine staining. Representative flow cytometry plots demonstrate the degree of IL-17 and IFNγ production from CD4+ T cells (gated on TCRβ+CD4+) in response to the stimuli  from the spleen (A). (B and D) Bar graphs show the frequency of IL-17 (B) and IFNγ positive cells in the spleen from the mice. Each data point represents an individual mouse in all of the bar graphs. (C and E) Supernatants were harvested at day 3 from the splenocyte (A) stimulation and the cytokines IL-17 (C) and IFNγ (E) were determined by ELISA. Data analysis was performed using ANOVA. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Data are representative of a single experiment.(A-E) Wild-type female mice were immunized one time with PBS (n = 5), Empty-GPs (n = 5), or 4X-SA-GP (n = 5). Five days after the vaccination, splenocytes from each mouse were stimulated with a mixture of all 4 (TIF)Click here for additional data file.S4 FigS. aureus proteins encapsulated in 4X-SA-GP by ELISA. The subclasses IgG1 (A) and IgG2c (B) were analyzed for specificity towards rClfA, rIsdA, rMntC, and SdrE. The read-out of the assay is the optical density (OD) at 450 nm for each serum sample. Each data point represents an individual mouse. Data analysis was performed using ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative of a single experiment.(A-B) Serum was collected from each group of vaccinated mice 2 weeks after one immunization with PBS, Empty-GP, or 4X-SA-GP (n = 5 mice per group). The serum was diluted 3 times at 1:1000, 1:10,000 and 1;100,000 and was then tested for antibodies specific for each of the 4 (TIF)Click here for additional data file.S5 Fig+ antibody or the corresponding isotype control antibody on day -1 and day 0. On day 0, all groups of the mice were infected i.p. with 2x107 CFUs of S. aureus (LAC USA300). (A) flow cytometry plots demonstrating the degree of CD4+ T cell depletion from pooled peripheral blood mononuclear cells (PBMCs) from each vaccination group of mice on day 0 before the mice were infected with S. aureus. (B) flow cytometry plots demonstrating the degree of CD4+ T cell depletion from pooled MLNs from each vaccination group of mice 24 hours after the mice were infected with S. aureus (day 1). Cells from (A) and (B) were gated on CD3+ cells.(A-B) Wild-type female mice were immunized once a week for 3 weeks with PBS (n = 9), Empty-GPs (n = 10), or 4X-SA-GP (n = 10). Four weeks after the final vaccination 4–5 mice per vaccination group were treated i.p. with anti-CD4(TIF)Click here for additional data file.S6 FigS. aureus proteins encapsulated in 4X-SA-GP by ELISA. The subclasses IgG1(A) and IgG2c (B) were analyzed for specificity towards rClfA, rIsdA, rMntC, and rSdrE. The read-out of the assay is the optical density (OD) at 450 nm for each serum sample. Each data point represents an individual mouse. Data analysis was performed using ANOVA. *p<0.05, **p<0.005. Data are representative of at least two experiments for serum at two weeks and a single experiment for serum at 8 weeks.(A-B) Two sets of wild-type female mice were immunized once a week for 3 weeks with PBS (n = 5), Empty-GPs (n = 5), or 4X-SA-GP (n = 5). Serum was collected from one set of mice  2 weeks after the final vaccination and 8 weeks after the final immunization for the other set of mice . The serum was diluted 3 times at 1:1000, 1:10,000 and 1;100,000 and was then tested for antibodies specific for each of the 4 (TIF)Click here for additional data file.


Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in humans. EspF is one of the best-characterized effector proteins secreted from the type three secretion system to hijack host cell functions. However, the crucial pathogen-host interactions and the basis for the intestinal barrier disruption during infections remain elusive. Our previous study screened and verified the interaction between host protein ANXA6 and EspF protein. Here, by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (CO-IP), we verified that EspF interacts with ANXA6 through its C-terminal domain. Furthermore, we found that both the constitutive expression of EspF or ANXA6 and the co-expression of EspF-ANXA6 could decrease the levels of tight junction (TJ) proteins ZO-1 and occludin, and disrupt the distribution of ZO-1. Moreover, we showed that EspF-ANXA6 activated myosin light chain kinase (MLCK), induced the phosphorylation of myosin light chain (MLC) and PKCα, and down-regulated the expression level of Calmodulin protein. Collectively, this study revealed a novel interaction between the host protein (ANXA6) and EspF. The binding of EspF to ANXA6 may perturb TJs in an MLCK-MLC-dependent manner, and thus may be involved in EHEC pathogenic function.Enterohemorrhagic  Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen; it has been linked to a broad spectrum of diseases ranging from bloody diarrhea to hemorrhagic colitis and even life-threatening hemolytic uremic syndrome (HUS)   after the colonization of EHEC/EPEC to IEC  co-precipitated with EspF , and mammalian expression plasmids encoding ANXA6 fused to the C-terminal of yellow fluorescent protein  . We inveHEK293 cell lines co-transfected with pECFP-N1 and pEYFP-C1 were set as a negative control (NC). Cells transfected with pECFP-YFP were a positive control (PC). Cells co-transfected with CE and YA were the experimental group (EX). The FRET efficiency of each group was assessed by fluorescence microscopy. The results showed that the average FRET efficiency of NC was 22.6, 56% for PC, and 74% for the experimental group. As shown in We employed the traditional CO-IP method to further validate the specific interaction domain of EspF with ANXA6. First, pECFP-EspF-Flag and pEYFP-ANXA6-HA were co- and single-transfected into HEK293 cells, and then the HEK293 cell lysate was obtained. We observed that EspF-Flag co-precipitated with ANXA6-HA. In contrast, interactions were not detected between IgG and ANXA6-HA . Thus, wThe N-terminal domain of the EspF protein plays a decisive role in EspF's targeting to mitochondria and nucleoli of host cells. In contrast, the C-terminal domain contains the primary protein-binding site with SNX9 and N-WASP  and pEYFP-ANXA6-HA (YA) were single- and co-transfected (CO) into Caco-2 cells. Cells transfected with plasmids pEGFP-N1 (E) were set as a blank control. The constitutive expression of EspF (EE) and ANXA6 (YA) depleted ZO-1 and occludin proteins. The co-transfected group decreased the level of ZO-1, but did not affect occludin. Meanwhile, the reduction effect on occludin protein was not as obvious as that on ZO-1 protein. Of the two proteins, ANXA6 appeared to have a stronger ability to reduce the level of ZO-1, while EspF was more efficient in depleting occludin .Between the monolayer Caco-2 cells, an “upper-like” complete and dense structure was formed, that is, TJs. The TJ protein ZO-1 is continuously distributed along the membrane under normal conditions. We transfected plasmids encoding EspF and ANXA6 proteins, and then observed the distribution of ZO-1 by immunofluorescence. As shown in espF and anxa6 to be expressed in equivalent amounts in the same cells.The T2A peptide is one of the most popular strategies used to express multiple genes in eukaryotic cells  was also lower than in the NC group .We then observed the localization of ZO-1 in Caco-2 cells transfected with EYEA. Non-transfected Caco-2 cells (NC) and cells expressing EYFP (EY) were used as controls. As shown in The data presented herein support the notion that EspF protein promotes the disruption of TJ integrity and accelerates the depletion of TJ proteins through the interaction with the host ANXA6 protein.in vivo and in vitro , and espF- complementation strain (p-ΔespF) (MOI = 100), and monitored TER over time. The TER value of uninfected (UI) group was stable. Six hours after infection, the TER value of the experimental group began to decline, and we observed a dramatic loss of TER in monolayers infected with WT at the 10th hour. In contrast, the ΔespF group eased the decline, whereas the TER value of the p-ΔespF group dropped sharply at the 10th hour, similar to the WT group , WT group . These despF, and p-Δ espF. Besides the UI group, we also set DH5α as a control group. Compared with the UI group, the level of ZO-1 and occludin decreased in the WT and Δ espF groups. Compared with the WT group, the level of ZO-1 and occludin was elevated in the Δ espF group  method to examine the interplay between EspF and host proteins, which provided specific candidates that interact with EspF  binds to actin and profilin, which may enable EspF to regulate actin polymerization, thereby interacting with ZO-1 and ZO-2 and resulting in TJ protein redistribution. EspF protein also cooperates with N-WASP and ARP2/3, inducing actin nucleation and disrupting paracellular permeability  pathogenicity island , espF-deficient strain (ΔespF), espF-complementation strain (p-ΔespF), and DH5α were preserved in our laboratory. Strains were routinely grown overnight at 37°C in Luria-Bertani (LB) broth with 0.1% kanamycin (ΔespF) or 0.4% chloramphenicol, and 0.1% L-arabinose (p-ΔespF). Before infection, strains were cultured in DMEM  with low glucose (1 g/L) to induce the expression of T3SS  supplemented with 10% fetal bovine serum  and 1 × penicillin-streptomycin solution (Gibco) under humidified 5% COespF and anxa6 constructs  and preserved in our laboratory. These two genes were cloned in different vectors to generate a label-tagged nstructs . Primer nstructs . PlasmidTo mimic the interaction of EspF protein and ANXA6 protein in cells, we used 2A self-cleaving peptide to construct a plasmid that can express EspF and ANXA6 in cells in equal amounts. Primers ET-F: CGGACTCAGATCTCGAGCTCAAGCTTCGATGCTTAATGGAATTAGTAA and ET-R: CCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCT CCCCTTTCTTCGATTGCTC were used to amplify EspF-T2A, and primers AT-F: TCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAT GGCCAAACCAGCACAGGG and AT-R: CCGGTGGATCCCGGGCC CGCGGTACCTTAGGCATAATCGGGTACAT were used to amplify T2A-ANXA6-HA. Then the PCR products of these two fragments were cloned into the HindIII/KpnI restriction sites of pEYFP-C1 to generate pEYFP-EspF-T2A-ANXA6-HA using the ClonExpress method . These constructs are listed in 5) were seeded on 6.5 mm Transwell chambers with a pore size of 0.4 μm  and cultured for 10–14 days. Transepithelial resistance was measured every other day using a voltmeter . The cell growth curve was drawn according to the TER value (Ω × cm2), and the rapid growth period of Caco-2 cells was determined. The cells were infected with WT, ΔespF, and p-ΔespF strains at their rapid growth period (multiplicity of infection of 100), and TER was measured three times for each well at the indicated time. The final TER values of each group were calculated by subtracting the resistance of the blank filter from the resistance of the filters containing cell lines and bacteria, then multiplied with the surface area of the 6.5 mm filters (0.33 cm2).Caco-2 cells  with DMEM growth medium without antibiotics. When the cells were ~60 to 70% confluent, p-ECFP-EspF-flag (1 μg) and/or pEYFP-ANXA6-HA (1 μg) were transfected into cells by Lipofectamine 3000 transfection reagent . Cells co-transfected with pECFP-N1 and pEYFP-C1 were set as a negative control (NC). Cells transfected with pECFP-YFP were a positive control (PC) as the distance between the encoded protein CFP and YFP was <10 nm.TM FV1000 software and is shown as the mean of each experiment; each experiment was repeated at least three times.For the FRET assay, fluorescent images were captured 48 h after transfection using a confocal microscope . The samples were excited at 440 nm, and the emission detection wavelengths were 520 ± 20 nm. The fluorescence images were captured under an appropriate filter set. Regions of interest were selected, and background subtraction was completed for all images. FRET efficiency was obtained with the “series analysis tool” of the Olympus FluoViewHEK293 cells over-expressing EspF and/or ANXA6 were prepared, as mentioned previously in the FRET assay. Non-transfected and transfected monolayers were washed with cold PBS. The cells were lysed in immunoprecipitation assay buffer  with 1 mM phenylmethanesulfonyl fluoride  for 30 min at 4°C. The cell lysates were harvested with centrifugation at 13,000 g for 15 min at 4°C, and the supernatant was removed and kept on ice. Subsequently, cell lysates were incubated with the primary antibody (anti-flag and anti-HA) with gentle rotation at 4°C for 3 h. Then, ~25 μL protein A+G Agarose beads  were added and incubated with the immunoprecipitation reaction at 4°C overnight. The beads were washed three times with radioimmunoprecipitation assay buffer (RIPA buffer). The protein was eluted with SDS-PAGE loading buffer  and boiled for 10 min, then subjected to SDS-PAGE and western blot analysis. The blots were probed with antibodies against Flag , HA , and actin .TM FV1000 software.To examine the effect of plasmid-encoded EspF and ANXA6 on the distribution of ZO-1, we seeded Caco-2 cells in glass-bottom cell culture dishes and transfected cells with pEGFP-EspF (1 μg) or/and pEYFP-ANXA6-HA (1 μg); pEGFP-N1 was set as an empty plasmid control. After 48 h from transfection, cell monolayers were washed with ice-cold PBS and fixed with chilled methanol for 5 min at room temperature (RT), then blocked with 10% ready-to-use goat serum for 30 min at RT. The fixed cells were then stained using primary antibody diluted with 10% goat serum  overnight at 4°C, followed by incubation with goat anti-rabbit secondary antibody  for 1 h. Then, the cells were stained for DNA using Hoechst  for 10 min at RT. The samples were visualized on an FV1000  confocal laser scanning microscope. The images captured were grouped, projected, and analyzed with Olympus FluoViewCaco-2 cells were seeded and transfected with plasmid pEYFP-EspF-T2A-ANXA6-HA as described above, to detect the effect of interaction between EspF protein and ANXA6 protein on the distribution of ZO-1 and check the localization of ANXA6 protein. The primary antibody anti-ANXA6  was used to stain ANXA6. Then, cells were stained for DNA and visualized as described above.The protein samples were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes , then blocked with 5% bovine serum albumin . Membranes were probed with anti-p-MLC , anti-MLC , anti-p-pkcα , anti-pkcα , anti-MLCK , anti-Calmodulin , and anti-actin , followed by horseradish-peroxi-dast-conjugated goat anti-rabbit IgG secondary antibody  or horseradish-peroxi-dast-conjugated goat anti-mouse IgG secondary antibody  for 1 h. The blots were detected using an ECL chemiluminescence substrates kit .P < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS 20.0 . The data are expressed as mean ± SD. Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by Dunnett's test for separate comparisons. The original contributions presented in the study are included in the article/YH and CW contributed to the design of the study. YH and JW carried out the majority of the experiments and statistical analyses. MF, JL, and XL contributed to the experimental work. BZ and WZ provided their expertise. All authors reviewed the draft and approved the submitted version.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.