Porphyromonas gingivalis produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl  and mono-P-tetraacyl  lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism.IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs. All of the Gram-negative bacteria studied so far have been shown to produce outer membrane vesicles (OMVs) via blebbing of the outer membrane (OM) that are then released as spheres ∼50 to 250 nm in diameter , 2 and s4–The Gram-negative bacterial envelope consists of an OM, an inner membrane (IM), and the periplasm , which is found in the periplasmic space between the two membranes. LPS is the main component of the outer leaflet of the OM, and a layer of phospholipids is present in the inner leaflet of the OM. Thus, the outermost layer of the OMVs contains predominantly LPS and phospholipids. OMVs also contain integral OM and OM-anchored lipoproteins and periplasmic proteins. Evidence from studies of OMVs from several bacteria shows that OMV production is a regulated mechanism since some components of the OM and periplasm are present in OMVs in amounts different from those in the cell –6, 9–11.–9–Although the levels of OMV production in Gram-negative bacteria can be affected by environmental factors or mutations in certain genes, non-OMV-producing strains have not been isolated , 12, sugPorphyromonas gingivalis is an important agent in the etiology of adult periodontal disease and produces several virulence factors that include extracellular cysteine proteases with specificities for Arg-X  and Lys-X  peptide bonds -α-d-Glcp-(1→4)-α-l-Rha-(1→3)-β-d-GalNAcp- are attached at position 2. One of the side chains contains Manα1→2Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Previous studies of P. gingivalis W50 have shown that there is selective packaging of proteins and virulence factors into the OMVs of P. gingivalis and certain abundant OM proteins are excluded  encodes an integral OM protein that appears enriched in the OMVs of P. gingivalis W50. Ishiguro et al.  are secreted into the extracellular milieu. Chen et al.  strain are very similar throughout the period tested (∼ 8 days), whereas the ΔPG0027 mutant strain shows a decrease in optical density (OD) after 72 h, indicating cell lysis; a typical growth curve is shown in PG0027 mutant strain is nonpigmenting (not shown). The growth curves of P. gingivalis W50 and the ΔPG0027 and CΔPG0027 strains were determined in duplicate, and the error bars are shown in PG0027 mutant strain was more susceptible to growth inhibition in the presence of Tween 20 (0.02%) than the parent and complemented strains , and W50 and the ΔPG0027 and CΔPG0027 strains were examined by transmission electron microscopy (TEM).P. gingivalis W50 and ΔPG0027 mutant cells grown in brain heart infusion (BHI) broth for 24 h. P. gingivalis W50  mutant strains  in the ΔPG0027 mutant strain compared to parent strain W50. The CΔPG0027 strain showed an increase in the yield of OMVs compared to the ΔPG0027 mutant strain, indicating that the trend was toward restoration of OMV formation by complementation. Low-resolution TEM images were subjected to visual inspection, and OMV formation was also determined by counting. The number of OMVs per 100 cells of the ΔPG0027 mutant strain was significantly reduced to a similar extent (∼50%) (P < 0.003) as that determined by weight per culture volume (The yields of OMVs (milligrams per 250 ml of culture) obtained from e volume  of parenPG0027 mutant strain, the CΔPG0027 strain, and the ΔPG902/PG0027 and ΔPG1711/PG0027 mutant strains .Synthesis of both O-LPS and A-LPS appears to be unaffected in the ΔP. gingivalis W50, the ΔPG0027 mutant strain, and the CΔPG0027 and CΔPG0027R strains by MALDI-TOF MS -3-OH-i C17:0 and one each of (R)-C16:0, (R)-3-OH-C16:0, and (R)-3-OH-i C15:0. Heterogeneity within these clusters reflects differences in fatty acid chain length (14 mass units), a phenomenon previously ascribed to the relaxed specificity of the fatty acyl chain transferase activity of P. gingivalis , mono-P-pentaacyl , and mono-P-tetraacyl  species; signals for non-P-pentaacyl  and non-P-tetraacyl  species were either very low or absent. Hence, rather than the reported loss of deacylation following inactivation of PG0027, these data suggest that PG0027 is required for lipid A dephosphorylation while the acylation status is unaltered by this mutation.Since it has previously been suggested that PG0027 is a lipid A deacylase , we examI-TOF MS . Average/z 1,372 . The clu species . Kumada  species  describengivalis . ConversCΔPG0027 strain showed clusters similar to those present in P. gingivalis W50 lipid A (rgpA promoter to induce expression (the CΔPG0027R strain), the results were dramatically different  and non-P-tetraacyl  species, were detected.The lipid A of the  lipid A . In the ifferent . In thisPG0027 into two strains in which the α-mannosidase genes PG902 and PG1711 (PG0027. The lipid A isolated from ΔPG902/PG0027 and ΔPG1711/PG0027 mutant strains (with an extra copy of PG0027 inserted) was also analyzed by MALDI-TOF MS , mono-P-pentaacyl , and mono-P-tetraacyl  species  and non-P-tetraacyl  species. However, lipid A from the P. gingivalis ΔPG902/PG0027 and ΔPG1711/PG0027 mutant strains showed a large increase in the signals for non-P-pentaacyl  species and a demonstrable increase in non-P-tetraacyl  species  and mono-P-triacyl  species. Signals for the nonphosphorylated species were very low or absent  and mono-P-tetraacyl species , whereas the dephosphorylation of lipid A species appears to be an active process that occurs between ∼8 h and 24 h of growth, as detected by MALDI-MS analysis .P. gingivalis ΔPG0027 mutant strain obtained by MALDI-TOF MS showed the absence of nonphosphorylated species, it was relevant to identify the position of the phosphate group in the mono-P-pentaacyl and mono-P-tetraacyl species of lipid A from the parent and mutant strains, i.e., whether it is a lipid A 1-phosphate or a lipid A 4′-phosphate. MALDI-TOF/TOF tandem MS (MS/MS) analyses of the lipid A cluster centered around the m/z 1,688 (mono-P-pentaacyl) and m/z 1,448 (mono-P-tetraacyl) species were performed. The results are shown in Since the profiles of the lipid A species of the m/z 1,688 from the P. gingivalis ΔPG0027 mutant strain , C-15(3-OH), phosphoric acid, and various combinations of these were observed as product ions in the TOF/TOF MS/MS spectrum of the mono-P-pentaacyl species of lipid A   were recovered in comparatively small amounts from P. gingivalis W50, it was not possible to perform MALDI-TOF/TOF MS/MS analysis of this peak. In the TOF/TOF MS/MS spectrum of the m/z 1,448 species of lipid A from P. gingivalis W50 and ΔPG0027, there were signals for the neutral losses of C-16(3-OH) and C-16 fatty acids, phosphoric acid, and fragment Z1 [lacking C-16(3-OH) and phosphoric acid]  and mono-P-tetraacyl  species of lipid A from both P. gingivalis W50 and the ΔPG0027 mutant strain carry the phosphate at C-1. Thus, we conclude that the phosphatase deficient in the ΔPG0027 mutant strain is lipid A 1-phosphatase.No cross-ring fragments were observed in the TOF/TOF MS/MS spectra of the lipid A structures of t strain  and m/z /z 1,448  from botz 1,688) . Since tic acid]  25). Si. Sim/z 1P. gingivalis W50, the ΔPG0027 mutant strain, and the CΔPG0027 and CΔPG0027R strains were measured with 4-nitrophenylphosphate as the substrate at 30°C at pHs between 7.4 and 8.3 in either discontinuous or continuous assays with a recording spectrophotometer. No phosphatase activity was present in the culture supernatants of P. gingivalis W50, the ΔPG0027 mutant strain, or the CΔPG0027 and CΔPG0027R strains, suggesting that this activity was not secreted into the culture medium by P. gingivalis.The results described so far have indicated that PG0027 has a role to play in the dephosphorylation of lipid A. Therefore, phosphatase activities in culture supernatants, intact whole cells, and sonicated supernatants of P. gingivalis W50 and the ΔPG0027 mutant and CΔPG0027R strains were measured at pHs 7.4 to 8.3, and activities at pHs 7.8 and 8.0 are shown in P. gingivalis ΔPG0027 mutant strain contains ≥90% of the phosphatase activity present in parent strain W50, indicating that the phosphatase activities in the sonicated supernatants of P. gingivalis W50 and the ΔPG0027 mutant strain are comparable.Phosphatase activities in sonicated cell supernatants  (periplasm plus membranes plus cytoplasm) of P. gingivalis W50 and the ΔPG0027 mutant strain grown for 24 h in BHI broth were measured with 4-nitrophenylphosphate concentrations of 0.25 to 4 mM, and the maximum velocities were determined with ENZFitter software. In this case, the assays are most likely a measure of the activities present in the periplasmic space/IMs. The phosphatase activities in intact cells of P. gingivalis W50 appear to show a pH maximum at 7.8 to 8.0, and the activities measured at these two pH values are shown . At pHs 7.4 and 8.3, the phosphatase activity of intact cells of the ΔPG0027 mutant strain were significantly lower (P < 0.03 at pH 7.4 and P < 0.02 at pH 8.3) than those of parent strain W50  expressed in E. coli  and the appearance of a bis-P-pentaacyl-peak at m/z 1,572, indicating that PagL shows lipid A 3-O-deacylase activity. However, membranes containing PG0027 expressed in E. coli do not alter the MALDI-TOF MS spectrum of Salmonella lipid A (O-)deacylate or dephosphorylate Salmonella LPS.Deacylase activity of PagL  shows signals for bis-P-pentaacyl, mono-P-pentaacyl, non-P-pentaacyl, and mono-P-tetraacyl species at m/z 1,772, 1,689, 1,570, and 1,448, respectively, and bis-P-hexaacyl species from E. coli lipid A at m/z 1,796 (E. coli membranes expressing PG0027 and PagL (not shown), the MALDI-TOF MS spectrum does not show major changes    identifiocarbons , is a moocarbons  describen et al. , and then et al. , 31, andn et al. , 32. The2+ and one for Mg2+, and are exposed to the environment. On that basis, it is therefore unlikely that PG0027 is a lipid A 1-phosphatase because its structure and location in the OM are not consistent with previous studies of this group of enzymes. Conversely, Hoopman et al.  shows similarity to class A bacterial nonspecific acid phosphatases (NSAPs)   and shown solved . The CTDn solved . HoweverE. coli by using the PagL deacylase of Salmonella expressed in E. coli as a control. PG0027 did not exhibit either lipid A O-deacylase or phosphatase activity against W50 lipid A or Salmonella lipid A, in contrast to the complete deacylation of Salmonella bis-P-hexa-acylated lipid A by PagL, in the assays. While we cannot rule out the possibility that the recombinant PG0027 protein was expressed in an inactive form, these data, together with the structural information given earlier, strongly suggest that PG0027 is not a lipid A-modifying enzyme. This suggests that PG0027 has an indirect effect on the lipid A 1-phosphatase activity in P. gingivalis. Thus, (i) lipid A 1-phosphatase activity in the P. gingivalis ΔPG0027 mutant strain is synthesized but not transported into the periplasm, (ii) the lipid A 1-phosphatase enzyme is synthesized and transported into the periplasmic space but is unable to hydrolyze the lipid A 1-phosphate because of the presence of a potent inhibitor/regulator of enzyme activity, or (iii) the lipid A 1-phosphatase enzyme is not synthesized in the ΔPG0027 mutant strain. The similar levels of total cellular phosphatase activity but differential levels in the periplasm in the parent and mutant strains suggest that the first two of these options are the most likely. Therefore, it is possible that PG0027 acts as a transporter or as an inhibitor/regulator of periplasmic lipid A 1-phosphatase activity in P. gingivalis W50.To experimentally address whether PG0027 acts as a lipid A 1-phosphatase as described above, we performed activity assays with recombinant PG0027 expressed in P. gingivalis are still controversial. Coats et al.  and outer (LPS) faces. The outer face of the OM can interact with cations in the milieu  because Pseudomonas aeruginosa, resistance cannot be detected until the tip of the AFM is ∼10 nm from the surface of an LPS micelle , used as a pressure-measuring device, has shown that for the isolated B-band LPS of  micelle , althougm the OM , 49. To The presence of nonphosphorylated species of lipid A in the OM may be beneficial to the organism by enabling it to avoid immune detection through Toll-like receptor 4 binding , 32 and P. gingivalis ΔPG0027 mutant strain, could stabilize the interactions between divalent cations and lipid A in the OM. Hence, lipid A dephosphorylation in P. gingivalis W50 is a regulated process caused by the ability of PG0027 to control the levels of phosphorylated and nonphosphorylated species of lipid A in A-LPS in the OM. The balance of these species has the potential to stabilize/destabilize the OM and hence influence the production of OMVs in this organism.The main nonbeneficial consequence of lipid A dephosphorylation, and hence the absence of negatively charged residues, is liable to be an inability to bind to divalent cations . Thus, the inability to bind divalent cations can potentially destabilize the OM. The absence of nonphosphorylated lipid A species, as in the case of the Chemicals were purchased from VWR, Lutterworth, Leicestershire, United Kingdom, or Sigma-Aldrich Co. Ltd., Poole, Dorset, United Kingdom, and were the purest grades available. Restriction and modification enzymes were purchased from New England BioLabs, and DNA purification reagents were obtained from Qiagen.P. gingivalis W50 and mutant strains were grown on blood agar plates containing 5% defibrinated horse blood or in BHI broth supplemented with hemin (5 μg ml−1) in an anaerobic atmosphere of 80% N2, 10% H2, and 10% CO2 (Don Whitely Scientific). Clindamycin (5 μg/ml) or tetracycline (1 μg/ml) was added when required. E. coli XL-1 Blue or XL-10 Gold (Stratagene) was used for plasmid maintenance and cloning. Antibiotics were added for cell selection  and plasmid selection .P. gingivalis mutant strains were performed as described previously  was monitored over a period of 8 days by measuring the OD at 540 nm (OD540).Construction and complementation of eviously  and are lsewhere . Growth P. gingivalis W50 and the ΔPG0027 mutant strain to Tween 20 was performed as follows. Appropriate amounts of 2% Tween 20 stock solution were added to BHI broth (10 ml) to yield BHI broth containing 0, 0.005, 0.01, and 0.02% Tween 20. One milliliter of cultures of P. gingivalis strains grown for 24 h was used as the inoculum. The OD540 was measured every 24 h.The susceptibility of 2) at 30°C. The assay consisted of 0.8 ml of buffer and 0.2 ml of 4-nitrophenylphosphate . Reactions were initiated by the addition of 50 μl of whole cultures (intact cells) or sonicated supernatants of P. gingivalis W50, the ΔPG0027 mutant, and the CΔPG0027 and CΔPG0027R strains, and the increase in absorbance was measured with a Beckman DU 800 spectrophotometer. The rates obtained were corrected for ionization of the 4-nitrophenol product when performing continuous assays. Phosphatase activities were also measured at pHs 7.4, 7.8, and 8.0 with 4-nitrophenylphosphate as the substrate at 30°C in a discontinuous assay. Reaction mixtures contained 2 ml of buffer and 0.5 ml of 4-nitrophenylphosphate  incubated at 30°C. The reaction was initiated by the addition of 50 μl of intact cells or sonicated supernatants of P. gingivalis W50 and the mutant and complemented strains. Aliquots (0.25 ml) were withdrawn at desired time intervals into 1-ml disposable cuvettes containing 0.75 ml of 0.75 M NaOH and mixed well, and the A405 was measured in a Beckman DU 800 spectrophotometer (Beckman Coulter) within 10 min. Each assay was done in triplicate, and mean values were used in calculations. Measurements of phosphatase activities in intact cells and sonicated supernatants were performed in triplicate as described above. Phosphatase activity was expressed as the change in A405 per minute per OD600 unit for intact cells or the change in A405 per milligram of protein for sonicated supernatants. Maximum rates and sK (substrate dissociation constant) values were obtained with ENZFITTER software . Standard deviations were calculated by using the values measured for each set of experiments  and are indicated in Alkaline phosphatase activity was measured in continuous assays at pHs 7.8, 8.0, and 8.3 –0.9% NaCl containing a complete cocktail of protease inhibitors (Roche) (buffer plus inhibitors). The washed cells were resuspended in a total of 2 ml of buffer plus inhibitors and sonicated with a small probe in a Soniprep Sonicator at a 12-μ amplitude for 5 × 30 s on ice. The sonicated samples were centrifuged in an Eppendorf centrifuge at 17,000 × g for 5 min at 4°C, and the supernatant was used in the measurement of phosphatase activities. Protein concentration was measured with the Bradford reagent (Bio-Rad) and bovine serum albumin as the standard.Cultures of g for 10 min at 22°C. Washing of lipid A enabled us to improve the quality of the MALDI-TOF spectra. The washed pellet remaining in the original tube was resuspended in 50 μl of ultrapure water and subjected to MALDI-TOF MS and gave clean spectra.Lipid A was isolated from freeze-dried whole bacterial cells (10 mg) and OMVs (5 to 10 mg) with TRIzol (Invitrogen) as described previously  and analMALDI-TOF MS was performed with a Bruker Microflex instrument fitted with a nitrogen laser operating at 337 nm with pulsed extraction in negative linear mode. Lipid A was analyzed with norharmane  at a concentration of 10 mg/ml in methanol-water  as the matrix as previously described .MALDI-TOF/TOF MS/MS was performed with an Autoflex TOF/TOF II  fitted with a nitrogen laser operating at 337 nm. Full-scale MS scan negative ions were analyzed in reflectron mode with norharmane (10 mg/ml in water plus 0.1% trifluoroacetic acid) as the matrix. The instrument was externally mass calibrated. For MS/MS, negative precursor ions were analyzed by LIFT and data were recorded with flexControl version 3.0 and processed with flexAnalysis version 3.0 at Durham University .P. gingivalis W50 and mutant strains grown in BHI broth for 24 h were centrifuged  at 26,000 × g and 4°C for 40 min, and the supernatant was filtered with a Nalgene 0.22-μm filtration apparatus. The filtrate was subjected to ultracentrifugation  at 180,000 × g and 8°C for 1 h. The pellet was resuspended in sterile phosphate-buffered saline, and the ultracentrifugation step was repeated, followed by resuspension of the pellet in water and freeze-drying. The yield of OMVs was taken as the dry weight in milligrams per 250 ml of culture.Volumes of 250 ml of cultures of P. gingivalis W50, the ΔPG0027 mutant strain, and the CΔPG0027 strain grown in BHI broth for 24 h were centrifuged in an Eppendorf centrifuge at 17,000 × g and 4°C for 15 min, and the cell pellets were used.Cultures of Specimens were examined in a JEOL JEM1230 electron microscope , and digital images were captured with an Olympus Morada camera.Imaging was performed with an FEI “Inspect” field emission scanning electron microscope , and secondary electron images were taken at 20 kV at a working distance of 10 mm.t test for paired values was used, and data were considered to be significant at a P value of <0.05.A Student


Then, PD-L1 and CD8A mRNA expression was analyzed using gastric cancer data from the TCGA database, confirming a positive correlation. Patient survival was assessed according to PD-L1 status and the T cell infiltration density. PD-L1 expression and a high density of CD8+ T cells in AGCs were associated with improved prognosis, whereas no significant difference was noted between PD-1 and CD3 expression. In contrast, a high density of FOXP3+ T cells in AGCs indicated a poor prognosis. Multivariate Cox regression analysis revealed that CD8+ T cell density acts as an independent predictor of overall survival (OS) in AGC patients. Taken together, this study further highlights targets for immune checkpoint-based therapy in AGC.Advanced gastric cancer (AGC) has high morbidity and mortality in East Asia, and it is urgent to explore new treatments to improve patient prognosis. Programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have exhibited remarkable activity in clinical trials and were approved by the FDA for clinical therapy in several types of tumors. Here, we evaluated PD-L1 expression and T cell infiltration in AGC. Positive tumor PD-L1 expression was detected in 171 AGCs (33.60%) out of 509 AGCs. PD-L1 expression was positively correlated with CD8 Lancet this year. The worldwide surveillance of cancer survival reported in this study demonstrated that GC ranked second in cancer incidence in China with lung cancer ranking first  [Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide . The recImmunotherapy, especially immune checkpoint blockade, has emerged as a promising cancer treatment . Immune PD-L1 expression in tumor cells and the immune checkpoint blockade therapeutic response have a close relationship. In a previous study involving 17 patients with PD-L1-negative tumors, none of the patients exhibited an objective response, while for PD-L1-positive tumors, 9 of 25 patients (36%) exhibited an objective response . In anotWe investigated PD-L1 expression and T cell infiltration in a tumor microarray (TMA) representing 509 AGC patients. The correlation between PD-L1 expression and T cell infiltration was examined in our study and using GC data from the TCGA database. Finally, the relationships between PD-L1 status and T cell infiltration with patient overall survival (OS) were analyzed.This is a retrospective analysis of 509 patients with primary gastric cancer who underwent gastrectomy at the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, from January 2006 to December 2011. The final follow-up date was December 31, 2017, for all cases examined. OS time was defined as the interval between the gastrectomy and patient death or survival. A total of 242 mortalities occurred, which were all due to cancer-associated causes. All patients received the standard treatments such as D2 radical resection and first-line adjuvant chemotherapy according to the NCCN guide. Only 25 patients did not finish the standard chemotherapy for their personal reasons or inability to tolerate side effects. There was no difference in the number of not finishing standard chemotherapy between PD-L1-positive and -negative groups. We excluded the following types of patients: (1) patients with recurrent gastric cancer after the radical operation, (2) patients receiving neoadjuvant chemotherapy or previous radiotherapy, (3) patients suffering from other malignant tumors, and (4) patients with autoimmune or immunodeficiency diseases.μm thickness.We collected formalin-fixed paraffin-embedded (FFPE) tissue blocks from the pathology department of Renji hospital. Tumor TNM stage was assigned based on pathological tumor, node, and metastasis staging per the American Joint Committee on Cancer (AJCC 8th edition) staging system. For each case, the diagnosis was confirmed by two senior pathologists through a review of H&E-stained slides. Representative FFPE blocks were chosen to punch onto glass slides to construct the TMA. Every patient's tumor tissue on the TMA was consecutive, and the TMA was constructed using a tissue arrayer with 5 This study was approved by the ethics committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine, for the use of samples. Informed consent was obtained from all enrolled patients before study inclusion.Immunohistochemistry (IHC) was performed on the TMA using antibodies specific to PD-L1 , PD-1 , CD3 , CD8 , and FOXP3 . Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes, and blocked with 10% BSA , slides were first incubated using the antibody at 4°C overnight and then labeled with the HRP second antibody  at room temperature for 1 h. Positive staining was visualized with DAB substrate liquid  and counterstained with hematoxylin . All the2) on the TMA for each case and counted the average CD3+, CD8+, and FOXP3+ cell density. According to the median number of stained cells , patients were dichotomized into the high and low density group. To evaluate Foxp3+ T cells, given that few cells were stained, we defined the high infiltration group as greater than 5 stained cells/HPF, whereas the remaining cases comprised the low infiltration group. Digital image analysis and Nikon DR-Si2 cell count software were used for the staining evaluation described above, and the results were verified by two senior pathologists who were blinded to the clinicopathological data.Tumor PD-L1 expression in the cytoplasm and membrane of tumor cells was evaluated based on immunostaining. The PD-L1-positive group was defined based on greater than 5% of stained cells regardless of cytoplasmic or membrane staining. The remaining cases comprised the negative group. The criterion for classification in the PD-1 high expression group was greater than 5 cells stained per high-power field (HPF), whereas the remaining cases comprised the low expression group . We chosFrom 450 GC samples from the TCGA database, we selected 415 tumor tissue samples except 35 normal tissue samples. We analyzed the correlation between PD-L1 and CD8A expression at the mRNA level.χ2 test or Fisher's exact test. The correlation between PD-L1 and CD8A mRNA expression was calculated using the Spearman correlation test. Survival analysis was performed using the Kaplan-Meier method and the long-rank test. Univariate and multivariate analysis were conducted using the Cox proportional hazards model to analyze prognostic factors. All statistical tests were 2-sided, and P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 16.0 statistical package software  or GraphPad Prism .Standard statistical tests were used to analyze clinical data. Associations between PD-L1 expression and clinicopathological factors were tested using a A retrospective cohort study of 509 AGC patients, including 49 TNM stage I cases, 172 TNM stage II cases, and 288 TNM stage III cases, was conducted. The median age of the AGC patients was 62 (22–89) years, and the median OS time was 48 (2–117) months. A total of 242 (47.54%) patients died during the follow-up period. Among the 509 cases, male patients (347/509) and a low position of the lesion (360/509) represented a large proportion of the cohort. In total, 96 and 80 cases exhibited blood vessel and perineuronal invasion, respectively, among the 509 AGC patients. The detailed clinicopathological characteristics of the patients are presented in P = 0.2255)  .P = 0.0045). No significant relationship was found between tumor PD-L1-positive status and other clinicopathological features.The relationship between tumor PD-L1 expression and the clinical characteristics of AGC patients is presented in +, CD8+, and FOXP3+ T cell infiltration in tumor tissues was evident (P = 0.0241). No close relationship between PD-1 expression and other clinicopathological features was found (Supplementary + T cell infiltration in tumor tissues was positively associated with the patient's Lauren type (P = 0.0243). CD8+ T cell infiltration in tumor tissues exhibited a close relationship with lymph node metastasis (P = 0.0242) . Significant differences in the number of PD-L1-positive patients were noted between the CD3+ and CD8+ T cell high and low infiltration groups . Regarding FOXP3, no significant difference in PD-L1-positive patients was noted between the high and low expression groups (P = 0.9215)  (No significant difference in the number of PD-L1-positive patients was noted between the PD-1 high and low expression groups ( 0.9215) . We consve group . Next, t 0.0001) .+ T cell infiltration were associated with improved OS compared with negative expression or the low infiltration group   (P = 0.3570 and P = 0.1092)  . No sign Figures .P = 0.005), high CD8+ T cell infiltration in tumor , and high FOXP3+ T cell infiltration in tumor  were revealed as protective or risk factors for OS in AGC patients. Next, we selected potential prognostic factors based on univariate results (P < 0.05) to conduct multivariable analysis. High CD8+ T cell infiltration in tumor , TNM stage , and length-diameter  could act independent predictors of OS for AGC patients. However, tumor PD-L1-positive status  and high FOXP3+ T cell infiltration in tumor  were not independent predictors for AGC prognosis. Other clinicopathological parameters, including blood vessel invasion , perineuronal invasion , and Lauren type  exhibited no significant differences in the multivariable analysis  demonstrated that high PD-L1 expression was associated with younger patient age and high tumor grade. No associations with sex, tumor size, stage, nodal status, EGFR, or KRAS mutation status were noted. PD-L1 expression appeared to be a favorable prognostic factor in early-stage disease, and the results differed for advanced-stage patients . In our + T cell infiltration in a pancancer analysis study. The results demonstrated that PD-L1 expression exhibited a positive correlation with CD8A expression. Patients exhibiting high or low expression of both proteins were increased compared with patients with high or low expression of only one protein [+ or CD8+ T cell infiltration given that the PD-L1 high expression group contained a larger percent of CD3+ or CD8+ T high infiltration patients than did the low expression group. GC analysis of the PD-L1 and CD8A mRNA expression levels reported in TCGA database revealed a positive correlation between PD-L1 and CD8A mRNA. Another study that classified melanoma tumors based on T cell infiltration and PD-L1 expression demonstrated that 38% were type I cancers (PD-L1+TIL+), 41% were type II cancers (PD-L1−TIL−), 1% were type III cancers (PD-L1+TIL−), and 20% were type IV cancers (PD-L1− TIL+) [+ T cell infiltration. This result was consistent with that of our study and another study of synovial sarcoma [High PD-L1 expression was associated with high CD8 protein . In our 1− TIL+) . PD-L1 eNext, we analyzed PD-L1 expression based on GC patient survival. Patients with high PD-L1 expression exhibited prolonged OS times compared with the low expression group. In fact, high PD-L1 expression was associated with a better prognosis than low PD-L1 expression in several cancer types, including gastric cancer , colorecγ when they encounter tumor antigens, causing an adaptive response to IFN-γ and leading to the upregulation of PD-L1 in tumor cells. The induced expression of PD-L1 in the tumor microenvironment creates a “shield” to avoid attack from activated effector T cells. Consequently, under these circumstances, PD-L1 expression is considered a marker of an active host antitumor immune response [Two mechanisms of the upregulation of PD-L1, including the innate immune response and adaptive immune response, have been proposed. The innate immune response leads to PD-L1 upregulation due to dysregulated oncogenic signaling pathways and chromosomal alterations and amplifications in the tumor. In the adaptive immune response, tumor-infiltrating cytotoxic T lymphocytes (CTLs) secrete IFN-response . It is n+ and CD8+ TILs remained independent prognostic factors in multivariate survival analysis [+ T cell tumor infiltrate indicated an improved prognosis compared with the low density group, and CD8+ T cell infiltration was an independent prognostic factor in the multivariate survival analysis. In CD3+ T cell infiltration analysis, no significant difference was noted between GC patients with high and low infiltration. It is possible that CD3+ T cells contained various T cell types with different functions in the immune response. CD8+ T cells act as a type of immune cell that directly kills or eliminate tumor cells in the tumor microenvironment. Conversely, FOXP3+ T cells can suppress antitumor immunity. Next, FOXP3+ T cell high and low tumor infiltration were analyzed based on OS. We found that a higher density of FOXP3+ T cell tumor infiltration was associated with a worse survival. This result was consistent with a previous study in other tumors [Based on the above analysis, PD-L1 expression associated with tumor-infiltrating immune cells was a positive prognostic feature. In a study to determine whether tumor-infiltrating lymphocytes (TILs) can predict the clinical prognosis in gastric cancer, the densities of CD3analysis . In our r tumors .+ and CD8+ T cell infiltration. PD-L1 and CD8A mRNA expression levels were positively correlated among GC patients in the TCGA database. Positive PD-L1 tumor expression and a high density of CD8+ T cells in AGCs were both associated with increased OS time, whereas no significant differences were noted in the PD-1 and CD3 high and low groups. In contrast, a high density of FOXP3+ T cell infiltration was associated with poor prognosis. Multivariate Cox regression analysis revealed that CD8+ T cell density could act as an independent predictor of OS in AGC patients. Taken together, positive tumor PD-L1 expression and high CD8+ T cell infiltration might have implications for targeting the PD-L1/PD-1 axis and the treatment of GCs. The prognostic value and immune pattern might be useful for guiding treatment in the future.In our study, we explored the relationship between PD-L1 expression and T cell tumor infiltration among 509 AGC patients. We found that PD-L1-positive status was correlated with high CD3


Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor. Agrobacterium-mediated genetic transformation is one of the most popular techniques used for the generation of transgenic plants  was used to transform Nicotiana benthamiana (N. benthamiana) plant cells in both transient assays and stable transformation. We demonstrated that the mutant strain GV2260 (recA-SacB/R) maintains its capacity of transforming plant cells, and its growth can be efficiently inhibited by regular tobacco tissue culture medium supplemented with 3% sucrose. Stable transgenic plants carrying a CRISPR-Cas9 construct for knocking out a WRKY transcription factor were successfully recovered after transformation with the mutant Agrobacterium strain. Therefore, the mutant A. tumefaciens strain should have great value for large scale, high-throughput plant transformation applications in the future.In this study, we attempted to integrate the Escherichia coli (E. coli) DH5α , and Escherichia coli (E. coli) helper P600  plants were propagated in a growth chamber programmed for 16 h light (140 μmol m−2s−1 cool white fluorescent irradiance) at 28°C and 8 h dark at 24°C Agrobacterium-mediated transient assays were conducted on three- to 4-week-old plants.recA gene was amplified from the genomic DNA of GV2260 using primers: recA For, 5′-caccatcgatcatgaagctcggt-3′ and recA Rev, 5′-gcgccggacttctcgacgat-3′. The PCR reaction as performed using the iProof™ high fidelity Taq DNA polymerase . The PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). The PCR product was separated on a 0.8% agarose gel, stained with 0.01% ethidium bromide solution, and visualized using the Gel-Document Image System™ under UV light (Bio-Rad).A 780 bp DNA fragment with deletion of the N and C-terminus of the recA and has been sequenced at the core facility of the Virginia Bioinformatics Institute .The PCR product was purified using the AccuPrep™ Gel Purification Kit  and cloned into the TopoEntr/D™ vector  following the instructions of the user manual. The derived plasmid vector was designated as TopoEntr-SacB/R and the ccdB gene cassettes. The Npt2 promoter-SacB/R fragment was amplified through overlap PCR from pEG101-SacB/R and pDSK519-GFP . The derived construct was designated as pLVC18L-Npt2-SacB/R. A ccdB gene cassette (frame B) (Invitrogen) was further cloned into the SmaI site of pLVC18L-Npt2-SacB/R to generate pLVC18L-Npt2-SacB/R-DesB. The recA gene fragment from TopoEntr-recA was subcloned into pLVC18L-Npt2-SacB/R-DesB using a Gateway®; LR cloning kit (Invitrogen) following the instructions of the user manual. The derived plasmid construct was named pLVC18L-Npt2-SacB/R-recA, and has been confirmed by sequencing at the core facility of the Virginia Bioinformatics Institute .The PCR product was cloned into the SacB/R-recA was integrated to the genome of A. tumefaciens strain GV2260 by tri-parental conjugation and was selected on LB medium supplemented with tetracycline (10 μg/mL) as previously described . To knock out NbWRKY70, we identified a guiding RNA (GCAATCGACGGGTTAATTCGCGG) targeting the NbWRKY70 gene. An Arabidopsis U6 promoter, NbWRKY70 guiding RNA, and the PAM terminator were amplified through overlap-PCR using primers 2284AtU6gRNA common For1, CAGCAACTCATTACAACTTGTTTaagctttcgttgaacaacgga; 2285AtU6gRNA common Rev1, CGACTCTAGACACGGGGTGGTTTaaaaaaagcaccgactcggtgcc; 2846NbWRKY70 gRNA For, GCAATCGACGGGTTAATTCGgttttagagctagaaatag; 2847NbWRKY70 gRNA Rev, CGAATTAACCCGTCGATTGCaatcactacttcgactcta.A putative tobacco WRKY transcription factor NbWRKY70 was identified from GenBank (accession number PmeI site of pM3UT-Cas9 using a Gibson cloning kit (New England BioLabs), where it contains a Cas9 gene driven by the Arabidopsis Ubiquitin 10 promoter. The Cas9 gene was originally codon optimized and synthesized based on the Arabidopsis genes . The derived construct was designated as pgRNA-NbWRKY70.The PCR product was gel purified and cloned into the NbWRKY70 were conjugated into A. tumefaciens strain GVV2260 and GV2260-SacB/R using tri-parental conjugation, selected on LB medium supplemented with rifampin 100 μg/mL, and Kanamycin 50 μg/mL or Spectinomycin 50 μg/mL as previously described  media supplemented with rifampicin 100 μg/mL, tetracycline 10 μg/mL, and kanamycin 50 μg/mL and incubated at 28°C for 2 days. Bacterial cells were harvested and re-suspended in induction buffer composed of 10 mM MgCl2, 10 mM MES (pH 5.6), and 100 μM acetosyringone and incubated for 3 h at room temperature. The bacterial inoculums were adjusted to OD600 nm = 0.6 and infiltrated into the stomata of the fully expanded N. benthamiana leaves using a 1-mL blunt-end syringe without a needle. The inoculated plants were incubated at room temperature under continuous light for 20–48 h before the detection of expressed proteins. The fluorescent signal of YFP-HA fusion protein was monitored 24 h after inoculation by fluorescent microscopy .Agrobacterium strain GV2260 or GV2260-SacB/R carrying plasmid pgRNA-NbWRKY70 were used for tobacco transformation following a previously described protocol  and NAA (0.1 mg/L) and 3% maltose at 25°C in the dark for 2 days. The infected leaf disks were soaked in liquid MS medium for 5 min and then rinsed one time with liquid MS medium. The leaf disks were then blotted dry and transferred to a selection medium . Each treatment has at least 100 leaf disks with three replicates.The selection mediums were incubated at 25°C under continuous light for 25–30 days for shoot regeneration. The transgenic shoots were transferred to rooting medium (MS medium supplemented with Kanamycin 100 mg/L and 3% sucrose).PmeI For, 5′-TGATAGAGTAGTTCATAT GGA-3′ and PMOA36-TBS PmeI Rev 5′-GCTTCC CAACCTTACCAGAG-3′. The PCR products were gel purified and sequenced at the core facility at Virginia Bioinformatics Institute.The putative transgenic tobacco plants were confirmed by PCR with primers: PMOA36-TBS Bacterial genomic DNAs were isolated using a ZR Fungal/Bacterial DNA MiniPrep™ . Plasmid DNAs were isolated using an AccuPrep™ Plasmid Extraction Kit . Plant genomic DNAs were isolated by using the CTAB method as previously described . The phenotype of transgenic plants will be further characterized in the future. Nevertheless, our result demonstrated that GV2260-SacB/R could successfully transform N. benthamiana plant cells to generate stable transgenic plants. It will be interesting to test the transformation capacity of GV2260-SacB/R in other plant species. In this study, we also confirmed that the CRISPR-Cas9 system is a powerful tool for introducing mutations on target genes in N. benthamiana (Nekrasov et al., After 2 months of culture selection, putative transgenic 0 Figure  because e Figure . To exame Figure . The PCRe Figure  (Li et ae Figure . The douAgrobacterium strain GV2260-SacB/R that is sensitive to sucrose. The mutant strain can be used for plant cell transformation as demonstrated by Agrobacterium-mediated transient assays and stable transformation. The overgrowth of mutant strain GV2260-SacB/R can be inhibited by 3–5% sucrose, a common carbon source used in plant tissue mediums. Therefore, GV2260-SacB/R can be a valuable tool for plant transformation research.We generated a mutant BZ conceived the project and the cloning strategy. YML and JM performed the experiments. ST, DK, YL, ZN, and XZ contributed vectors and other reagents. YML, JM, ST, DK, YL, ZN, XZ, ZL, and BZ analyzed the data and wrote the draft manuscript. YML and BZ wrote the final manuscript. All authors read and approved the final manuscript.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.


Escherichia coli O157:H7 (EHEC O157:H7) causes hemorrhagic colitis and the formation of characteristic attaching and effacing (A/E) lesions in humans. Given the severe sequelae of EHEC O157:H7 infection, it is critical to develop effective vaccines for human use. However, for achieving this goal many hurdles need to be addressed, such as the type or subset of antigens, adjuvant, and the delivery route. We developed a candidate vaccine by inserting the bivalent antigen espA-Tir-M composed of espA and the Tir central domain into Lactobacillus acidophilus. The recombinant L. acidophilus (LA-ET) was safe in a cell model and excluded EHEC O157:H7 from LoVo cells at rates of nearly 94 and 60% in exclusion and competition assays, respectively. LA-ET inhibited the induction of A/E lesions by EHEC O157:H7 cells in vitro. Oral immunization with LA-ET induced higher levels of specific mucosal and systemic antibody responses in mice. Moreover, LA-ET enhanced interferon-γ and interleukin-4 and -10 production, which was associated with mixed helper T (Th1/Th2) cell responses, and protected against EHEC O157:H7 colonization and infection in mice at a rate of 80%. Histopathological analyses revealed that orally administered LA-ET reduced or inhibited A/E lesions and toxin-induced systemic injury. These findings demonstrate that LA-ET induces both humoral and cellular immune responses in mice and is therefore a promising vaccine against EHEC O157:H7 infection.Enterohemorrhagic  Escherichia coli O157:H7 (EHEC O157:H7) is a zoonotic enteric pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans  proteins, and surface fimbrial and afimbrial adhesions . The T3SLactobacillus acidophilus (L. acidophilus), Lactococcus lactis, and Bifidobacterium have been given a “generally regarded as safe” (GRAS) designation and are widely used in human food production. Additionally, LAB’s are considered as a useful tool for the development of novel oral vectors and have largely replaced attenuated pathogens with mucosal delivery strategies , which is composed of espA and the Tir central domain (Tir-M), into L. acidophilus. We evaluated the specific immune responses elicited in mice and protection against EHEC O157:H7 challenge both in vitro and in vivo.Lactic acid bacteria (LAB) such as rategies . Moreoverategies . Vaccinerategies . Lactocoifficile . Howeverunctions . To dateLactobacillus acidophilus ATCC4356  was cultured in de Man, Rogosa, and Sharpe (MRS) agar or broth at 37°C in an atmosphere of 5% CO2. EHEC O157:H7 strain EDL933 (our laboratory stock) was grown at 37°C in Luria–Bertani (LB) broth supplemented with 5 g/l streptomycin. The pMG36e plasmid (our laboratory stock) was maintained in DH5α cells and cultured in LB broth with 200 μg/ml erythromycin, and pMG36e-based plasmids were maintained in MRS agar or broth with 0.1 μg/ml erythromycin. The LoVo human colonic cancer cell line (our laboratory stock) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2.E. coli O157:H7 EDL933 using primer pairs with the following sequences: P1 (forward), 5′-A AAA CTGCAG GAT GGA TAC ATC AAA TGC A- 3′ and P2 (reverse), 5′-GCT ACC TCC TCC ACT TCC GCC TCC TTT ACC AAG GGA TAT TGC TG-3′ for espA; P3 (forward), 5′-GGA GGC GGA AGT GGA GGA GGT AGC AGC CCA ACC ACG ACC GAC-3′ and P4 (reverse), 5′-CCC AAGCTT TTA GGC TTG CTG TTT GGC CTC TT-3′ for Tir-M; and P1/P4 for espA-Tir M. Restriction enzyme sites (PstI and HindIII) are underlined. The fusion gene espA-Tir-M was inserted into plasmid pMG36e.To construct the recombinant plasmid pMG36e-espA-Tir-M, the espA-Tir-M (ET) fragment was amplified by overlap extension PCR from espA (GenBank accession no. KJ549678.1) and Tir-M (GenBank accession no. NC002655.2). EspA and Tir-M fragments were joined with a linker (GGA GGC GGA AGT GGA GGA GGT AGC). Both espA and Tir-M were PCR-amplified from L. acidophilus cells according to the manufacturer’s instructions for the expression system in Lactococcus lactis  with appropriate adjustment. Briefly, cells were cultured in 4 ml of MRS broth with 0.05% cysteine-HCl medium at 37°C for 48 h. When the optical density at 600 nm (OD600) reached 0.6, 4 ml of culture was diluted in 100 ml of MRS broth with 0.5 M sucrose and 0.05% cysteine-HCl, followed by incubation for approximately 24 h until the OD600 was 0.8. The culture was cooled for 10 min and centrifuged at 5,000 rpm for 20 min at 4°C, then washed twice in 20 ml of 0.5 M sucrose buffer. The cells were re-suspended in 5 ml of pre-cooled transformation buffer composed of 10 mM ammonium and 0.5 M sucrose (pH = 6.0), then centrifuged at 5,000 rpm for 20 min at 4°C and re-suspended in 400 μl of transformation buffer. The recombinant plasmid was transformed into L. acidophilus cells by electroporation using a Pulse Controller apparatus  at 2.5 kV and 25 μF. Transformed bacteria were re-suspended in MRS broth and cultured at 37°C for 1 h, and were plated on MRS agar  with 0.1 μg/ml erythromycin at 37°C for 48 h. Positive colonies of transformed bacteria were identified by PCR-amplifying the target gene (espA-Tir-M) and sequencing. The empty vector pMG36e transformed into L. acidophilus cells (LA) served as a control.The recombinant plasmid pMG36e-espA-Tir-M was transformed into L. acidophilus (LA-ET) was cultured in MRS broth with 0.1 μg/ml erythromycin at 37°C for 24 h. The supernatant was precipitated with trichloroacetic acid followed by two washes with acetone. A 20 μl volume of sterile distilled water was added to dissolve the supernatant proteins, which were separated by 12% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blotting using an anti-EHEC O157:H7 polyclonal antibody  according to the manufacturer’s instructions.The recombinant LA-ET or LA at a multiplicity of infection (MOI) of 500:1 was incubated with LoVo cells for 12 h, after which these cells were harvested and washed with PBS. The cells were re-suspended in 500 μl of binding buffer containing 5 μl of FITC-Annexin V and 5 μl of PI. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. Finally, the cells were detected using flow cytometry according to the manufacturer’s instructions.5 LoVo cells per well were cultured in 24-well plates. For the exclusion assay, the LoVo cells were pre-inoculated with LA-ET or LA (MOI = 500:1) for 90 min and then, EHEC O157:H7 (MOI = 100:1) was added for co-incubation for 90 min at 37°C. For the competition assay, LA-ET or LA and EHEC O157:H7 were added to LoVo cells, followed by incubation at 37°C for 3 h. LoVo cells inoculated with EHEC O157:H7 (MOI = 100: 1) for 90 min served as a positive control. To measure the total number of adherent EHEC O157:H7 bacteria, cells were lysed and the cell suspension was plated in triplicate on LB agar and incubated for 24 h at 37°C, after which the number of colony-forming units (CFUs) was counted. Each experiment was repeated three times. Results were calculated as a relative adhesion percentage [100 × /(number of adherent bacteria in the positive control group)].Inhibition of EHEC O157:H7 by LA-ET by the colonized LoVo cells was evaluated using the following assays as previously described . Briefly2. After incubation, cells were washed three times with PBS (pH 7.4) and fixed in 4% paraformaldehyde in PBS (pH 7.0) for 10 min. Following a 30 s wash in PBS, cells were permeabilized in 0.5% Triton X-100 in PBS for 5 min. The cells were washed once and then 200 μl of 100 nM rhodamine phalloidin  was added and incubated in the dark for 30 min. The cells were washed three times and the DNA was counterstained for 30 s with 200 μl of 100 nM DAPI in PBS. The samples were examined with a confocal laser scanning microscope.Fluorescence actin staining (FAS) was performed to detect the formation of A/E lesions. Briefly, LoVo cells were pre-incubated with LA-ET (MOI = 500:1) for 3 h at 37°C and then EHEC O157:H7 (MOI = 100:1) cells were added for co-incubation for 3 h at 37°C in 5% COn = 15): PBS, LA and LA-ET. The PBS group was orally administered with 100 μl sterile PBS; the LA group was orally administered with 1 × 109 CFU LA; and the LA-ET group was orally administered with 1 × 109 CFU LA-ET. All mice were immunized on days 0, 3, 7, 10, 21, and 24 (the day of first immunization regarded as 0 day) and serum samples were collected on days 0, 7, 21, and 35 and stored at -80°C until use. Fecal extracts were collected on day 35 for sIgA measurement by enzyme-linked immunosorbent assay (ELISA) as previously described . All experimental and animal handling procedures were approved by the Institution Animal Care Committee of Southern Medical University (permit no. 44002100006397) and were performed in accordance with the approved relevant guidelines. The mice were sacrificed by cervical dislocation at the end of the experiment. LA-ET strains were grown as described above, then collected and washed twice with sterile PBS. Mice were randomly divided into the following three groups .Total serum IgG  and fecal IgA were measured by indirect ELISA as previously described . ELISA pn = 10) received streptomycin (5 g/l) in the drinking water from 3 days before infection until the end of the experiment to clear intestinal flora and to enhance EHEC O157:H7 colonization (n = 5) was not treated with streptomycin. Mice were challenged by oral inoculation of 1 × 1010 CFU of EHEC O157:H7 in PBS. Fecal shedding of EHEC O157:H7 was monitored at 2-day intervals as previously described , which indicates that the LA-ET-expressed espA-Tir-M fusion protein was mainly a secreted protein. The secretion of espA-Tir-M was confirmed by reaction with the anti-EHEC O157:H7 polyclonal antibody with western blotting .The espA-Tir-M fusion gene was amplified by overlap extension PCR, yielding the expected 816-bp product, which was visualized by agarose gel electrophoresis (data not shown). The fragment was cloned into the pMG36e vector, and the recombinant pMG36e-espA-Tir-M plasmid was transformed into Figure 2).Comparisons of the cytotoxic effect in LoVo cells showed no obvious difference in the LA-ET group compared to the normal group and LA group (P = 0.000). For the latter, the relative adhesion was 35.8% ± 6.83% for LA-ET and 39.4% ± 8.26% for LA (P = 0.501), and both LA-ET and LA excluded EHEC O157 from LoVo cells by about 60% (P = 0.000) .The ability of LA-ET to interfere with EHEC O157:H7 adhesion to LoVo intestinal epithelial cells was investigated using exclusion and competition assays. For the former, both LA-ET and LA excluded EHEC O157 from LoVo cells at a rate of almost 94% . By contrast, cells pretreated with the LA-ET no longer demonstrated EHEC O157:H7 adhesion and A/E lesions . The LoVo cells  and LoVo cells incubated with LA-ET  were normal.Fluorescent-labeled phalloidin staining of F-actin in LoVo intestinal epithelial cells showed that EHEC O157:H7 challenge of LoVo cells induced bacterial attachment to cells (green arrows), effaced microvilli, and recruited F-actin into pedestals to form A/E lesions (white arrows) . In addition, serum antibody titer increased after each immunization with LA-ET , reaching a peak on day 35 (P = 0.000). sIgA was also measured in fecal samples . Consistent with systemic antibody responses, sIgA levels were increased in mice immunized with LA-ET (0.26 ± 0.08) as compared to control mice (P = 0.000).Significant IgG responses were elicited in mice immunized with LA-ET as compared to controls  . Serum IL-10 level was higher in the LA-ET  and LA  groups than in the PBS group (51.4 ± 19.5) . Moreover, the serum IFN-γ level was higher in the LA-ET group (102.2 ± 63.0) than in the control LA  and PBS  groups .Cytokines play an important role as part of the immune response against infections. Serum IL-4 concentrations were significantly higher in the LA-ET group , whereas 60% of mice in the LA group died between days 1 and 5. Clinical symptoms included weight loss, hunched posture and lumbering gait. The post-infection survival data revealed that the protection rate was 80% in LA-ET.The protection efficiency of LA-ET was evaluated based on survival, fecal shedding, and histopathology. Within the streptomycin-treated immunization groups, 90% of mice were susceptible to EHEC O157:H7 challenge and died between days 1 and 3 post-infection in the PBS control group (P = 0.000); one vaccinated mouse had stopped shedding on day 9, and all mice were negative by day 13. In contrast, control mice shed bacteria throughout the 15-day time course .In addition, the duration of fecal shedding shown by the LA-ET group was significantly shorter than that in the LA group (P = 0.000). Additionally, the shorter duration of fecal shedding shown by the LA group in comparison to the PBS group was significant (P = 0.007) .No mice died in the non-streptomycin-treated immunization groups when challenged with EHEC O157:H7. Comparisons of the durations of EHEC O157:H7 fecal shedding showed a significantly shorter duration in the LA-ET group (8.2 ± 0.89 days) compared to the LA and PBS groups  .The intestine was dissected out from the mice sacrificed 15 days after challenge. The pathological changes of non-immunized infected mice included major intestinal injury  accompanied by damage to the kidney (hyperemia of mesenchyme capillaries), liver (mesenchyme hyperemia), and spleen (splenic sinusoid dilation and ecchymosis) (data not shown). However, the colon of immunized mice infected with EHEC O157:H7 appeared normal, and these animals produced well-formed stools with no obvious mucosal thickening, similar to non-infected mice (Escherichia coli O157:H7 is a major cause of gastroenteritis. Most infected individuals develop HUS, which is prevalent in children (Enterohemorrhagic children . EHEC O1children . It has children . Probiotchildren .in vitro but do not affect EHEC O157:H7 adhesion to host cells (In this study, LA and LA-ET were both shown to exclude EHEC O157 from LoVo cells in exclusion and competition assays, which highlights the important role of probiotics in interrupting progressive infection with EHEC O157:H7. This finding was consistent with studies in which probiotics reduced intestinal infection . Furtherst cells . TherefoSalmonella induced significant specific IgG in serum and sIgA in feces to protect mice from a challenge of EHEC O157:H7 (LA-ET used for oral immunization can prevent gastric infection and allows direct contact between the antigen and the immune system. Significant IgG and sIgA responses were elicited in mice immunized with LA-ET and protective effects were also confirmed. A previous study showed that attenuated EIS-producing recombinant  O157:H7 . These f O157:H7 . The maj O157:H7 . We obse O157:H7 .L. acidophilus strains play an important role in protecting mice against EHEC O157 infection and may be useful vaccine carriers for combating enteric pathogens in humans.Reduced intestinal colonization is regarded as an important criterion for protection against EHEC O157:H7 infection. Oral immunization of mice with LA-ET resulted in a reduction of EHEC O157:H7 fecal shedding in two infection models. The infection model under the oral streptomycin treatment condition emphasized the protective effects of humoral and cellular immune responses induced by LA-ET. LA-ET blocks adhesion of EHEC O157:H7 to intestinal tract, which may be associated with the sIgA. The sIgA antibodies play an important role in blocking the attachment of EHEC O157:H7 to epithelial cells . The infin vivo cannot be measured. In addition, the expression level of the antigen in LAB is weak and the enzymes in the gastrointestinal tract may digest the proteins (L. lactis strain expressing the EHEC antigen EspB and this strain expressed rather low levels of antigen EspB. However, they optimized the expression of EspB in L. lactis through secretion of EspB either under constitutive or nisin-inducible control, and the strains successfully induced immune responses (L. acidophilus expressing the EHEC O157:H7 antigen. Thus, the optimal condition for LA-ET that can enhance stimulation of strong immune responses still needs to be explored. High expression of L. lactis vector is promising for vaccine development.The histopathology indicates that administration of LA-ET has the ability to reduce or inhibit A/E lesions, which suggests that LA-ET could reduce the colonization of EHEC O157:H7 in the intestinal tract and inhibit A/E lesions. Moreover, the mechanism utilized by LA-ET against EHEC O157:H7 is different from that of antibiotics. LA-ET was safe in a cell model, but EHEC O157:H7 released toxins when treated with antibiotics, which could cause toxin-induced systemic injury. Thus, LA-ET is a promising candidate vaccine against EHEC O157:H7, especially for children and the elderly. Although oral immunization with LA-ET induced protective immunity, an important deficiency should be noted. First, the antigen dose provided by LA-ET proteins , which mesponses . Collectesponses . In our in vitro, and oral immunization strain induced both humoral and cellular immune responses in vivo and protected against EHEC O157:H7 colonization and infection in mice. These findings suggest that oral LA-ET is a promising candidate vaccine against EHEC O157:H7 infection.LA-ET was capable of inhibiting A/E lesions and bacterial adhesion RL did the experiments with gene construction, cells and mice, analyzed data, and wrote the manuscript. YZ did the experiments with mice and analyzed data. BL designed the experiments and contributed to revising the manuscript. YL and YW did the experiments with gene construction and mice. SD and BZ did the experiments with mice. XW and HF provided overall directions and contributed to revising the manuscript.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.


While it is known that the heart remodels progressively with age, data characterizing the relationship between cardiac remodelling and function on physical function among the aged are limited.We investigated the association between left ventricular (LV) concentricity and LV function with handgrip strength  and timed-up-and-go test (for lower extremity function) among clinically asymptomatic elderly.0.67 (mass/end-diastolic volume0.67) and LV function by resting tissue Doppler imaging (TDI) performed at the septal and lateral mitral annulus, deriving myocardial systolic velocity (S), diastolic velocity (E) and ratio of E/A.In this community-based cohort of 28 patients free of known cardiac disease (mean age 73.8 ± 4) with preserved LV ejection fraction (mean LVEF 69.8 ± 6.4) and cardiac index (mean 3.1 ± 0.57), we assessed cardiac remodelling by cardiac MRI , lateral S , septal A , and between timed-up-and-go and LV concentricity , lateral E/A , septal A . By regression analysis, LV concentricity  (with adjustment for systolic blood pressure) and lateral E/A  were independently predictive of handgrip strength and timed-up-and-go respectively.These preliminary observations provide important insights into a possible link between subclinical alterations in cardiac structure and function and physical function, further study is required to clarify these findings with a view to preserve health and function amongst the elderly.


N-glycolylated gangliosides and other self-antigens. This antibody is able to induce an anti-idiotypic IgG response and B-T idiotypic cascade, even in the absence of any adjuvant or carrier protein. P3 mAb immunization induces the expression of activation markers in a significant percentage of B-1a cells in vivo. Interestingly, transfer of both B-1a and B-2 to BALB/Xid mice was required to recover anti-P3 IgG response in this model. In fact, P3 mAb activated B-2 cells, in vitro, inducing secretion of IFN-γ and IL-4, although this activation was not detected ex vivo. Interestingly, naïve CD8+ T cells increased the expression of activation markers and IFN-γ secretion in the presence of B-1a cells isolated from P3 mAb-immunized mice, even without in vitro restimulation. In contrast, B-2 cells were able to stimulate CD8+ T cells only if P3 was added in vitro. Using bioinformatics, a MHC class I-binding peptide from P3 VH region was identified. P3 mAb was able to induce a specific CTL response in vivo against cells presenting this peptide. Both humoral and CTL anti-idiotypic responses could be mechanisms to protect against the self-reactive antibody, contributing to keeping the tolerance to self-antigens.P3 is a murine, germline, IgM mAb that recognizes  N-glycolylated gangliosides and sulfatides, both self-antigens in mice Y[YF]C[AT]RSGV was presented in five (0.07%) antibodies , four of which expressed the YYCARSGV sequence. Finally, we wanted to evaluate whether the predicted peptide was able to mediate an in vivo cytotoxic T lymphocyte response after P3 mAb immunization. We used a CTL in vivo assay where BALB/c mice immunized once with P3 mAb or the control IgM received splenic cells labeled with CFSE and loaded or not with the MYYCARSGV peptide. The result showed that P3 mAb immunization was able to generate a specific CTL response that significantly kills only the transferred splenic cells loaded with the specific peptide (+ T cell cytotoxic response in the syngeneic model against an idiopeptide.Since B cells activated with P3 mAb were able to activate CD8 peptide . Thus, PN-glycolylated gangliosides, but it also recognizes other self-molecules like sulfatides [P3 is a monoclonal antibody, generated in BALB/c mice , that halfatides . In addilfatides  and othelfatides . Furtherlfatides , which ilfatides , and is lfatides , phospholfatides , 55, chrlfatides , and carlfatides . The strlfatides –12. Studlfatides , 58–62.γ and IL-4, but not IL-10, generating a mixed Th1/Th2 pattern, in congruence with the mixed IgG subtypes detected in anti-P3 mAb response. However, this activation was not confirmed in vivo, since we could not detect an increase in the percentage of B-2 cell overexpressing the activation markers directly ex vivo, after P3 immunization.In order to elucidate which B cell populations were participating in the anti-P3 idiotype response, BALB/Xid mice were immunized with P3 mAb. No IgG response was detected against P3 mAb, pointing to an important role of B-1a cells in the idiotypic response induced by this antibody. In fact, P3 mAb was able to activate this population in vivo, increasing the percentage of B-1a cells overexpressing CD25, CD69, and CD86. Our previous study showed that P3 was able to recognize up to 19% percentage of B-1a cells. This high percentage suggests a clonotypic recognition, where several B-1a clones are involved, which can be explained by the presence of a regulatory idiotope on P3 variable region . Further+ T cells were required to generate the anti-P3 mAb IgG response in BALB/c mice, suggesting an important role for these cells at the first stages of the anti-idiotypic response [+ cytolytic activity with the capacity to stimulate or inhibit B cell responses [+ T cells can either directly activate B cells to secrete antibodies [+ T cells, in the presence of peritoneal B-1a or splenic B-2 cells. In both cases, CD8+ T cells showed an increase in activation markers, as well as a higher production of effector molecules and IFN-γ. However, there are differences in the capacity of these two B cell populations to activate CD8+ T cells. The activation of the B-1a cells in vivo by P3 mAb immunization was enough to enable B-1a cells to activate naïve CD8+ T cells in vitro. B-1a cells isolated from P3 mAb-immunized mice stimulated naïve CD8+ T cells, even without adding P3 mAb to the culture. These results suggest the activation levels achieved by B-1a cells in vivo, along with the fact that a high percentage of B-1a cells recognized by P3 mAb [+ T cells in vitro. On the other hand, despite being isolated from P3-immunized mice, B-2 cells required further in vitro stimulation with this antibody in order to be able to properly activate CD8+ T cells. This difference with B-1a cells might be explained by two facts: first, there is a lower percentage of splenic B-2 cells recognized by P3 mAb, and therefore, their representation in the in vitro culture is much lower than that for B-1a cells. Second, contrary to B-1a cells, when we tested B-2 cell activation ex vivo, we did not see an increase in the expression of activation markers. Thus, B-2 cells need to be restimulated in vitro to induce CD8+ T cell activation. Based on the fact that B-2 cells need to be restimulated in vivo in order to activate CD8+ T cells, while B-1 cells can induce this activation directly ex vivo, without further stimulation, we hypothesize that B-1a cells should be the population presenting the idiopeptide to CD8+ T cells, in vivo, during the generation of the anti-P3 idiotypic response.It was demonstrated in the previous work that the immunization with P3 mAb induced not only B cell but also T cell activation , 77. We response . Severalesponses –31. Sometibodies  or inhiby P3 mAb , are eno+ T cells in vivo, since it has been shown that CD8+ T cells can be activated by self-immunoglobulins and be cytotoxic against the B cell-secreting antibodies against a self-molecule [H region of P3 mAb. T cells are tolerant in a large extent to germline-encoded Ig V-region sequences [H. Usually, the available idiotype-specific T cell repertoire is largely confined to T cells that recognize rare idiopeptides due to somatic mutations or N region additions [H was MYYCARSGV, located between FR3 and CDR3, a region very rich in N-additions [+ T cells, which was located in the NH2-terminal VH region, before FR1 [+ T lymphocytes in vivo, demonstrated that the immunization with P3 mAb generated a specific CTL response against cells presenting the peptide.Next, we studied whether P3 was also able to activate cytotoxic CD8molecule , 78–80. equences , which idditions . In our dditions . This lofore FR1 . The pep+ T cells, against anti-self antibody-secreting cells, in the control of autoimmunity. In fact, it has been demonstrated that idiotype-reactive T lymphocytes inhibit the development of autoimmune interstitial nephritis in rats [H region, codes 001716 [Several reports have shown the activation of suppressive idiotype-specific T cells in the response to self-antigens –86, sugg in rats , as well in rats , 86. Ints 001716  and AF27s 001716 , were isEvaluation of B cell reconstitution and functionality after transfer to BALB/Xid mice.


LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated  In coRecently, the presence of G-quadruplexes in viruses and their involvement in viral key steps have also been reported . G-quadrin vivo, eventually resulting in defective cellular immunity, and thus leading to full-blown AIDS. The Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates that there were 35.3 million people living with HIV at the end of 2012 compared with 26.2 million in 1999 – a 35% increase. Although important progress has been achieved in preventing new HIV infections and prolonging HIV patients’ life with antiretroviral therapy, the infection cannot be eradicated and therefore AIDS-related illnesses are projected to continue as a significant worldwide cause of premature mortality in the coming decades if a decisive cure is not found  from the promoter region of the HIV-1 LTR.Several G-quadruplex ligands have been developed against cellular G-quadruplexes implicated in tumor pathogenesis; at least two molecules have also been tested and proved effective as antiviral agents against HIV ,44. ReceTaq polymerase stop assay and in the construction/mutagenesis of the plasmids for the luciferase reporter assay were purchased from Sigma Aldrich . All other DNA oligonucleotides were purchased from Integrated DNA Technologies  or chemically synthesized on an ABI 394 DNA/RNA synthesizer. Synthesized oligonucleotides were purified and dialyzed successively against 20 mM KCl solution and water before being lyophilized. DNA concentration was expressed in strand molarity using a nearest-neighbor approximation for the absorption coefficients of the unfolded species , run in TBE buffer for 3 h at 80 W, and finally visualized by phosphorimaging (Typhoon FLA 9000).The molecular size of G-quadruplexes was visualized by non-denaturing polyacrylamide gel electrophoresis (PAGE). Samples were incubated in 10 mM potassium phosphate buffer (pH 7) before loaded on 20% polyacrylamide gels with 40% (v/v) sucrose added before loading. Gels were run with 10 mM potassium phosphate buffer (pH 7) at room temperature for 90 min at 120 volts. DNA migration was imaged using UV-shadowing. For Circular dichroism (CD) spectra were recorded on a JASCO-815 spectropolarimeter using 1-cm path length quartz cuvettes. DNA samples  were dissolved in a buffer containing 70 mM potassium chloride and 20 mM potassium phosphate (pH 7) in a reaction volume of 500 μL. For each experiment, an average of three scans was taken, the spectrum of the buffer was subtracted, and the data were zero-corrected at 320 nm.LTR-IV G-quadruplex was monitored by UV absorption on a JASCO V-650 UV-Vis spectrophotometer. DNA concentration was 5 μM. Samples contained 70 mM potassium chloride and 20 mM potassium phosphate, pH 7. Absorbance was monitored at 295 nm and zero-corrected at 320 nm, over the temperature range from 20 to 90°C. Melting curves were baseline-corrected to compute the fraction of folded G-quadruplex over varying temperature. The melting temperature was determined as the temperature at which half the population was in the folded state.The thermal denaturing of the 15N labeling (2H labeling (13C-1H)-HMBC and (13C-1H)-HSQC as previously described  were dissolved in a buffer containing 70 mM potassium chloride and 20 mM potassium phosphate (pH 7). Resonances for guanine residues were assigned unambiguously by using site-specific low-enrichment labeling , site-splabeling  and throlabeling . Spectraescribed . Inter-pescribed .LTR-IV G-quadruplex were first calculated by performing distance geometry simulated annealing using the XPLOR-NIH program  and 2H2O . The glycosidic dihedral restraints were based on intra-residue NOEs of H1′-H6/8 cross-peak intensities observed at the mixing time of 100 ms.Structures of the  program . The ten program . Prior tescribed . FinallyLTR-IV G-quadruplex have been deposited in the Protein Data Bank under accession code 2N4Y.The coordinates of the WT) HIV-1 LTR region (corresponding to segment −381/+83 in the HIV-1 genome) was inserted into the promoterless luciferase reporter vector pGL4.10-Luc2 , as previously reported (G18T mutant pGL4.10-Luc2/LTR vector was generated using QuikChange mutagenesis kit (Stratagene/Agilent Technologies) with the following primers: PrMut G18T (i) 5′-CCTGGGCGGGACTGGGGATTGGCGAGCCCTCAGATCC-3′ and PrMut G18T (ii) 5′-GGATCTGAGGGCTCGCCAATCCCCAGTCCCGCCCAGG-3′, where the mutated base is shown in boldface and underlined. Both the WT and G18T LTR sequences were confirmed by sequencing. Plasmid size was confirmed by Hind III linearization on 1% agarose gel.The wild-type (reported . The G18WT and G18T mutant LTRs was assessed in human embryonic kidney 293T (HEK293T) cells seeded in 12-well plates (2 × 105 cells/well). Cells were transfected 24 h post-seeding with pGL4.10-LTR-WT or pGL4.10-LTR-G18T (100 ng/well) using TransIT-293 transfection reagent , according to the manufacturer's protocol. After 30 min, cells were treated with increasing concentrations (1–4 μM) of BRACO-19  for 24 h. Luciferase activity was measured using the britelite plus Reporter Gene Assay System  in a Victor X2 multilabel plate reader . Cells were lysed in lysis buffer  and protein concentration was determined by bicinchoninic acid (BCA) assay . Luciferase signals were subsequently normalized to total protein content, according to the manufacturer's protocol . All experiments were performed twice and in duplicate.Luciferase activity of the HEK 293T cells sustain all viral steps with the exception of virion attachment and entry as they lack cell receptors recognized by HIV-1. They can be transfected with the HIV-1 proviral genome to produce fully infectious viral particles, indicating that their cytoplasmic/nuclear protein makeup is capable of sustaining viral transcription and replication .Taq polymerase stop assay was carried out as previously described (WT Taq 5′-TTTTTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGTTTTTCTGCATATAAGCAGCTGCTTTTTGCC-3′ or LTR-II+III+IV G18T Taq 5′-TTTTTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGATTGGTTTTTCTGCATATAAGCAGCTGCTTTTTGCC-3′) in lithium cacodylate buffer in the presence/absence of 100 mM KCl by heating to 95°C for 5 min and gradually cooling to room temperature. Where specified, samples were incubated with BRACO-19 (62.5–250 nM). Primer extension was conducted with 2 U of AmpliTaq Gold DNA polymerase  at 37°C for 30 min. The marker sample was obtained by treating the elongation WT product with the sequencing protocol of Maxam et al.  using Jalview . The analyzed sequences belong to a broad spectrum of HIV-1 subtypes, both non-recombinant subtypes of Group M and circulating recombinant forms.Conservation of the G18 base involved in the LTR-IV sequence displays 12 well-resolved peaks in the imino proton region  – corresponding to a single central G-tetrad layer. A single transition was observed in the thermal denaturing curve of LTR-IV  showing a melting temperature of 50.5°C in ∼100 mM K+. Collectively, these biophysical data suggest that LTR-IV predominantly forms a major G-quadruplex species that adopts a monomeric parallel-stranded conformation.The NMR spectrum of the n Figure , indicatn Figure , is indiLTR-IV sequence. Guanine imino (H1) protons were unambiguously assigned for all guanines by carrying out 15N-filtered experiments of sequences containing site-specific 4% 15N enrichment . Assignment of selected aromatic and methyl protons of thymine residues was made by through-bond correlation experiments, using site-specific 4% 13C,15N labeled samples  Thes Figure . (ii) ThLTR-IV sequence. Specifically, we examined the role of the T19 bulge and the ability of the G-quadruplex to form using other combinations of guanine bases. We synthesized a series of sequences containing base substitutions and/or base deletions  suggesting these mutations are highly disruptive to the native conformer. Collectively, these data demonstrated the T19-bulge is not mandatory for the formation of a parallel G-quadruplex from the LTR-IV region of the HIV-1 LTR. In contrast, the guanines in the G14-G16 and G18-G21 stretches are required.Mutated sequences were analyzed using NMR to ascertain their ability to form G-quadruplex and to gauge the folding similarity based on their imino proton chemical shift pattern Figure . The modLTR-IV G-quadruplex, a G18-to-T (G18T) mutation was introduced in the full-length HIV-1 LTR promoter fused to a luciferase reporter gene. As the G18T mutation was shown to abolish the LTR-IV G-quadruplex  and mutant G18T LTR-Luc plasmids were transfected into HEK 293T cells and the luciferase activity was measured. The promoter activity of the mutant G18T LTR was significantly reduced to 23% of the activity of the WT LTR . We found a 99.5% degree of conservation for G18. There were five sequences observed to contain G18 mutations, four sequences contained a T mutation and one sequence an A at position G18 (Supplementary Tables S1 and 2). Collectively, these data support the crucial role of this position in virus replication and suggest that the LTR-IV G-quadruplex could act as a modulator of activity in the HIV-1 LTR promoter.Considering the dramatic effects of incorporating a single LTR-IV G-quadruplex adopts a well-defined parallel-stranded G-quadruplex containing a T-bulge  We have shown here that when the capacity for LTR-IV to form a G-quadruplex is lacking (by G18T mutation), the LTR promoter activity is highly silenced in cells  Ins Figure  and D.LTR-IV G-quadruplex forms less readily than the LTR-III G-quadruplex, with a low population of the LTR-IV G-quadruplex forming in the full-length promoter  would likely lead to increased transcriptional activity. A possible negative implication of over-active transcription could be premature cell death prior to completing virion production. Alternatively, over-active transcription could have ramifications on viral latency.We demonstrate that the r Figure  and D. SLTR-III and LTR-IV G-quadruplex formation may act as a regulator of the viral promoter. The rational and selective targeting of the LTR-IV structure could lead to sharp effects on HIV-1 replication. Understanding the structures of LTR G-quadruplexes is a valuable first step in the rational design of a selective small molecule to target this region of the viral genome and modulate the transcriptional behavior of HIV.We propose the balance of LTR-IV G-quadruplex formed in the LTR promoter of proviral HIV-1 genome. Biophysical data show that LTR-IV adopts a monomeric and parallel-stranded G-quadruplex in physiological solution. In the LTR-IV G-quadruplex, a single-nucleotide bulge interacts with a nearby single-nucleotide loop in a distinctive manner not reported previously. Conservation analysis shows these residues are highly conserved in viable HIV-1 strains. We suggest the loop-bulge interaction observed in LTR-IV is a promising feature that could be exploited in the design of molecules to specifically target this structure. The LTR-IV G-quadruplex structure presented here is valuable in understanding both the biology of the HIV-1 proviral LTR and its potential as a therapeutic target.This work reports the NMR solution structure of the PDB ID: 2N4Y.