In the present study, we aimed to investigate their placental transcript synthesis, labor associated differential expression and localization at feto-maternal interface, and their functional implication in spontaneous labor. The study involved using feto-maternal interface  from two groups of healthy pregnant women at term (≥37 weeks of gestation), undergoing either elective C-section with no labor , or normal vaginal delivery with spontaneous labor . The immune function of SP-D, on term placental explants, was analyzed for cytokine profile using multiplexed cytokine array. SP-A, SP-D and MBL transcripts were observed in the term placenta. The ‘SLv’ group showed significant up-regulation of SP-D (p = 0.001), and down-regulation of SP-A (p = 0.005), transcripts and protein compared to the ‘NLc’ group. Significant increase in 43 kDa and 50 kDa SP-D forms in placental and decidual tissues was associated with the spontaneous labor (p<0.05). In addition, the MMP-9-cleaved form of SP-D (25 kDa) was significantly higher in the placentae of ‘SLv’ group compared to the ‘NLc’ group (p = 0.002). Labor associated cytokines IL-1α, IL-1β, IL-6, IL-8, IL-10, TNF-α and MCP-1 showed significant increase (p<0.05) in a dose dependent manner in the placental explants treated with nSP-D and rhSP-D. In conclusion, the study emphasizes that SP-A and SP-D proteins associate with the spontaneous labor and SP-D plausibly contributes to the pro-inflammatory immune milieu of feto-maternal tissues.Collectins, collagen-containing Ca Immunological components are an essential part of mammalian pregnancy due to semi-allogenicity of the fetus. Fetal tolerance is considered to be induced via an immunological cross-talk by the spatial and temporal expression of pro-inflammatory and anti-inflammatory molecules in the gestational tissues 2+ dependent C-type lectins, are secretory proteins integral to immunomodulation and host defense. Members of this family include surfactant protein A (SP-A), surfactant protein D (SP-D) and mannan binding lectin (MBL), which share the human chromosomal location between 10q11.2–10q23.1 Collectins, collagen-containing Cath to 8th week gestational villous and extra villous trophoblast At extra-pulmonary locations, synthesis of SP-A, SP-D and MBL, has been reported in the human lower female reproductive tract and these proteins are implicated in protection against reproductive tract infections A progressive increase in the levels of SP-A, SP-D and MBL proteins has been reported in human amniotic fluid (AF) with advancing gestation Conspicuously, AF has been anticipated as the major source for SP-A, SP-D and MBL proteins in placenta. Thus, we investigated placental synthesis, labor associated differential expression and localization of the collectins at feto-maternal interface, and their functional implication in spontaneous labor.Term feto-maternal tissues were obtained from two groups of participants, five from women undergoing Caesarean section with no labor  and five from women undergoing normal vaginal delivery with spontaneous labor  at the Department of Obstetrics and Gynaecology, Seth G.S. Medical College and KEM Hospital, Parel, Mumbai, with written informed consent obtained prior to the sample collection. Comparison of these two study groups involves one more variable (mode of delivery), besides onset of labor, and different modes of delivery may affect placental mRNA and protein expression profiles. The study design excluded the participants undergoing normal labor with caesarean section owing to association with more variables. The study was approved by the Institutional Ethics Committee for Clinical Research (152/2009-NIRRH), NIRRH (ICMR) and Ethics Committee for Research on Human Subjects (EC/GOVT-7/2009), Seth G.S. Medical College and KEM Hospital, Parel, Mumbai. All the women were in the normal fertility period (20–40 years), and had singleton pregnancy , with normal child birth weight. Medically, no reproductive tract infections, chronic diseases, or pathophysiological conditions of pregnancy were observed or reported by the participants and they did not receive any hormone therapy. The gestation matched feto-maternal tissue samples were collected in sterile PBS/saline in both groups.The collected placental cotyledon was dissected under stereomicroscope for the feto-maternal tissues. The separated placental and decidua basalis tissues were used for further analysis in the study. Hematoxylin and Eosin staining was performed to examine the morphology of decidual and chorionic villi, and cytokeratin 7 staining was used to distinguish the isolated trophoblasts from decidual tissues in both the study groups (data not shown). The placental or decidual samples were partly (a) suspended in 10% neutral formaldehyde (NBF) for immunohistochemical analysis, (b) stored at −80°C in trizol for real time RT-PCR analysis, and (c) stored at −80°Co in lysis buffer for Western blot analysis.Human term amniotic fluid was collected and pooled from women at term undergoing C-section (n = 20) at the Department of Obstetrics and Gynaecology, Seth G.S. Medical College and KEM Hospital, Parel, Mumbai, with written informed consent from the participants obtained prior to the sample collection. The pooled amniotic fluid was stored at −20°C until further use for protein purification. The study was approved by the Institutional Ethics Committee for Clinical Research (153/2009-NIRRH), NIRRH (ICMR) and Ethics Committee for Research on Human Subjects (EC/GOVT-6/2009-KEM), Department of Obstetrics and Gynaecology, Seth G.S. Medical College and KEM Hospital, Mumbai.2 and pH 7.4. Pre-equilibrated maltosyl-agarose beads with TCB , were added to supernatant in 20 ml/L ratio and the pH was readjusted to 7.4. The SP-D and maltose-agarose binding was carried out at room temperature for 2 h with mild shaking. Maltosyl-agarose beads were washed extensively with TCB, and 1 M and 0.5 M NaCl in TCB to remove any non-specific interactions. Elution of SP-D proteins was performed by 150 mM MnCl2 (in 20 mM Tris pH 7.4) at room temperature for 2 h or overnight at 4°C on shaker. The tubes were centrifuged and supernatant containing purified SP-D was collected. Rest of the SP-D strongly associated with the beads was eluted with 20 mM EDTA in 20 mM Tris-HCl, pH 7.4 at room temperature with mild shaking for 1 h. Supernatant collected after MnCl2 elution contained SP-D proteins only, while EDTA eluted fractions contained both SP-A and SP-D proteins and they were further separated by gel filtration and analyzed by silver staining  and Western blot respectively . The purified nSP-D protein was assayed for endotoxin content (given below), its binding and ability to agglutinate A. fumigatus conidia and its potential to enhance killing of fungal conidia by human PBMCs in vitroThe SP-D protein was purified with modifications in the protocol reported earlier using reagents sourced from Sigma, USA Escherichia coli as described earlier The recombinant fragment of human SP-D containing neck and CRD domains and a part of collagen domain (rhSP-D) was expressed under the bacteriophage T7 promoter as inclusion bodies in The placental total RNA was isolated by the trizol method and 3 µg was reverse transcribed using Superscript III first strand synthesis kit . 1–3 µl of the cDNA was used for each 25 µl real time PCR reaction using SYBR green . Primers were custom-made by Sigma Oligos . Details about the primer pair sequences, annealing temperatures and product size is given in the t standard method after normalizing with the 18S rRNA abundance in the same samples. Melting curve analysis was conducted to determine the specificity of the amplified products and to ensure the absence of primer-dimer formation. All the products yielded the predicted melting temperature and product size on the gel. All the analyses were performed in triplicates and the mean value was taken for further calculations.Real time RT-PCR was carried out with a hot start at 95°C for 3 min and amplified at 95°C–30 sec; 56°C–30 sec; 72°C–30 sec; for 40 cycles and final extension at 72°C for seven minutes on Biorad-iCycler . The semi-quantitation of the transcripts was carried out using ΔΔC2O2 in methanol, and antigens were retrieved by boiling the slides in sodium citrate buffer . The tissue sections were blocked in 5% normal goat serum  for 1 h at room temperature in antibody diluents  and probed overnight with mouse monoclonal antibody to SP-A  (1: 20), polyclonal SP-A antibody raised in goat  (1: 100), mouse monoclonal SP-D antibody  (1: 50), or mouse monoclonal antibody to MBL  (1: 50) in antibody diluent at 4°C. The kits for immunohistochemistry  were applied further as prescribed by the manufacturer. Tissue sections were incubated with biotinylated secondary antibody reagent for 2 h and signals were enhanced using avidin conjugated HP for 30 min at room temperature. For polyclonal goat antibody to SP-A, secondary anti goat IgG raised in donkey, conjugated with HRP  (1: 250) was used for 3 h in moist chamber at room temperature. The sections were incubated with 3-amino-9-ethylcarbazole (AEC) substrate for 10 min in dark and counter stained with hematoxylin  for 3-5 minutes. For the negative controls, tissue sections were treated with antibody diluent alone instead of the primary antibody. Slides were mounted using D.P.X mountant  and observed under the microscope. All the samples for each individual antibody were processed using the same protocol and at the same time. Photomicrographs were taken with an Axioplan microscope . The immunoperoxidase staining was quantified using NIS Elements Imaging Software ver 4.10 . Expression levels of collectins were calculated by multiplication of the positive area and mean optical intensity Serial placental or decidual tissue sections of 5 µm thickness were collected on the poly-L-lysine coated slides. Sections were deparafinized in xylene and gradually rehydrated with 90%, 70%, 50%, 30% and distilled water each for 5 min. Endogenous peroxidase activity was blocked by 3% HThe placental or decidual (∼100 mg) tissue was minced and homogenized in 0.5 ml of lysis buffer containing 8 M urea (Sigma) and 2% CHAPS  supplemented with protease inhibitor cocktail and nucleases . Samples were vortexed for 1 h followed by centrifugation at 13,000 rpm for 40 min at 4°C. Protein concentration in the supernatants was estimated using the Micro BCA kit . A total of 30 µg placental protein was loaded in each lane and resolved on 10% SDS-PAGE, using reagents obtained from Sigma unless mentioned. Proteins were transferred onto nitrocellulose membrane  and membranes were blocked in 5% non-fat dry milk in TBS overnight at 4°C. The excess of milk protein was removed by washing the blot with washing buffer (TBS-0.002% TritonX-100). Blot was probed with mouse monoclonal antibody to SP-A  (1: 150), mouse monoclonal antibody to SP-D  (1∶150), mouse monoclonal antibody to MBL  (1∶250), and polyclonal anti SP-A raised in goat , (1∶1000), in TBS containing 1% nonfat dry milk. The polyclonal β-actin antibody raised in rabbit  (1∶2000) was used for normalization. Polyclonal anti-mouse IgG raised in goat conjugated with HRP  (1: 1000) or anti-rabbit IgG antibody raised in goat linked with HRP  (1: 1000) or anti-goat IgG raised in donkey conjugated with HRP  (1: 5000) in TBS containing 1% nonfat dry milk were used and signals were detected using femto-level chemi-luminiscence detection method . In view of the presence of several activated proteases, including MMP9, in term placenta undergoing labor and ability of MMP9 to proteolytically cleave SP-D, we evaluated the MMP9 cleaved SP-D product in the term placenta of the two groups using monoclonal antibody to SP-D  (1∶150) 2, 37°C for six hours. The culture medium and explants were recovered for further analysis of cytokines and transcripts. The levels of cytokines secreted were evaluated using the multiplex cytokine array comprising of twelve cytokines . The mean value of each cytokine was obtained for every sample and presented as mean ±SEM.The placental tissue explants (∼100 mg) from C-section (‘NLc’ group) were prepared within half an hour of surgery and cultured in the DMEM/F12  and supplemented with 1% antibiotics and 10% FBS  in presence or absence of either purified human SP-D (nSP-D) or purified recombinant fragment of human SP-D (rhSP-D) dissolved in PBS. The placental explants (n = 3) in triplicate were treated with PBS (control) or 0.5, 1.0 and 5 µg/ml of nSP-D/rhSP-D in a 24 well culture plate. The culture plates were incubated at 5% COThe MTT  assay was used to evaluate the effect of nSP-D/rhSP-D proteins on the viability of placental explants. The culture medium of treated or untreated placental explants was removed after 6 h incubation, and the explants were washed with sterile PBS. The placental explants were transferred to the 0.25 mg/ml MTT solution in sterile PBS and incubated for 2 h. The freeze-killed placental tissue was used as negative control in the experiments. At the end of incubation, the MTT solution was drained and acidified isopropanol (0.02N HCl) was added to extract the formazan produced by viable cells in the tissue explants. Absorbance of extracted formazan was measured at 540 nm, normalized with absorbance of the freeze-killed explants and plotted as percentage of the control . The expThe relative quantitation was evaluated by one way ANOVA using MS Excel or GraphPad, and significance was set at p<0.05.Fig. S1). The immunoblot analysis further confirmed the presence of SP-A, SP-D and MBL proteins in the term placenta. Interestingly, the monoclonal antibody to SP-A, identified two major protein bands at ∼38 kDa and ∼46 kDa in the placenta, besides a ∼34 kDa minor protein band , while the human term amniotic fluid showed two bands of SP-A at 28–34 kDa and ∼50 kDa. The monoclonal antibody to SP-D identified two reported Fig.S2b). The monomeric form of serum MBL protein of 32 kDa, was present in the human term amniotic fluid and placenta . The synthesis of SP-A, SP-D and MBL and presence of proteins in term placenta prompted us to investigate their expression during the spontaneous labor.The transcript analysis showed the synthesis of SP-A (both SP-A1 and SP-A2), SP-D and MBL in the human term placenta (p>0.05), was observed in the placental tissues of ‘SLv’ group compared to the ‘NLc’ group [The differential expression analysis of the SP-A, SP-D and MBL transcripts was carried out in term placental tissues of the spontaneous labor group (‘SLv’) and placenta from C-sectionno labor (‘NLc’) groups. Transcript analysis revealed a significant up-regulation of the SP-D  in the placental tissues of ‘SLv’ group compared to the ‘NLc’ group Fig.1A. The quantitative analyses of SP-A, SP-D and MBL proteins were carried out in placental tissues of both the groups. Similar to the transcript expression data, we found a significant decrease in ∼38 kDa and ∼46 kDa protein bands of SP-A by 2 fold (p = 0.004) and 1.4 fold (p = 0.006) respectively in the placenta of ‘SLv’ group compared to the ‘NLc’ group [Immunohistochemistry was used to evaluate the localization of SP-A, SP-D and MBL proteins in the placental and decidual tissues of both groups. The mesenchymal stromal cells of the chorionic villi from both groups were intensely stained for SP-A protein Fig. 2a.p<0.05) and decidual tissues (p<0.05) of the ‘SLv’ group compared to the ‘NLc’ group  compared to ’ ‘NLc’ group (‘DNLc’) [c’ group . Similarc’ group Fig. 2f.p<0.05) and decidua (p<0.05) of ‘SLv’ group compared to the ‘NLc’ group . In the Western blot analysis of decidua, similar to the placenta, significant increase in both the isomers of SP-D protein was observed in the ‘SLv’ group, with more prominent increase in 43 kDa (p = 0.002) than 50 kDa isomer (p = 0.004) . We asked if the levels of SP-D are altered in placental tissues due to MMPs during spontaneous labor. The 25 kDa SP-D fragment was increased in the placental tissues of the ‘SLv’ group compared to the ‘NLc’ group .Matrix metalloproteinases, involved in collagenolysis, are the key molecules up-regulated during labor. SP-D is known to be cleaved by MMP9 in vitro immune effects of nSP-D or rhSP-D on placental explants of the ‘NLc’ group were evaluated. The placental explants were treated with 0.5, 1.0 and 5.0 µg/ml concentration of either nSP-D or rhSP-D and PBS treated explants were used as controls. Both nSP-D and rhSP-D induced the pro-inflammatory cytokine secretion by placental explants in a dose dependent manner .To get an insight into the relevance of increased SP-D levels in the placenta, the t manner Fig. 5A.We report the synthesis of transcripts of three members of the collectin family: SP-A, SP-D and MBL, by the human term placenta and their expression and localization at the feto-maternal interface during spontaneous labor. Comparative analysis of the placental and decidual tissues from the study participants of two groups ‘spontaneous labor’ (‘SLv’ group) and ‘no labor’ (‘NLc’ group) suggested significant up-regulation of expression of SP-D and down-regulation of SP-A during spontaneous labor.−/−, SP-D−/− and SP-A/D−/− mice delivered at term during the first pregnancy. However, a significant delay in the parturition was observed during second pregnancy in SP-A/D−/− mice and was attributed to decrease in production of IL-1α and IL-6 in the myometrial tissue This study, for the first time, showed association of a significant increase in the SP-D at the feto-maternal interface with spontaneous labor. An important role of SP-D in the parturition process was evident by significant association of the Met31Th SP-D gene polymorphism in the fetus, but not in mothers, with the spontaneous preterm birth in Finnish population et al., we observed the presence of SP-A transcript as well as protein in the term trophoblasts of both study groups The labor associated decrease of SP-A in the decidual tissues is in coherence with a recent report that SP-A was highly expressed in the decidual (vimentin-positive) cells from tissues collected before labor and was significantly reduced in the tissues collected after labor −6 M), but inhibited at the higher concentration (10−5M) Expression of the three collectins is differentially regulated by different transcription factors even though they are located in close vicinity of each other (human chromosome region (10q11.2–10q23.1), and have sequential similarity. The TTF-1 (thyroid transcription factor-1) is essential for expression of SP-A in the lung cells 2O2 decreases SP-A, and increases SP-D transcripts via MAPK/JAK-STAT pathway in human lung cells. Glucocorticoids show a similar effect through specificity protein-1 (SP-1) in fetal lung explants culture Oxidative stress, an important factor during pregnancy, could affect the expression of anti-oxidant SP-A and SP-D molecules at the feto-maternal interface −/− mice show seven fold elevated levels of SP-D, suggesting that the two genes regulate each other via feedback mechanisms Further, this paradoxical regulation of SP-A and SP-D expression could be due to the decreased stability of SP-A mRNA in presence of higher concentrations of glucocorticoids Various forms of SP-A and SP-D protein were observed in the present study  as reported previously −3 µM) and rhSP-D (1 µg∼16.7×10−3 µM). SP-D has been implicated with both anti-inflammatory and pro-inflammatory roles. In the presence of LPS, SP-D induced pro-inflammatory NF-κB pathway in macrophages mediated by the interaction of its collagen domain with calreticulin/CD91complex In vivo and in vitro nitrosylation of the two cysteine residues at the N-terminus results in formation of S-nitrosothiol (SNO)-SP-D and disassembling of the oligomeric structure of SP-D. SNO-SP-D shows increased chemotaxis for macrophages and induced p38 phosphorylation and MAPK activation mediated by binding to calreticulin/CD91 complex Increase in pro-inflammatory immune response in gestational tissues during normal delivery contributes to cervical tissue ripening and decidual reaction leading to labor Observations from this study for occurrence of collectins in placenta are consistent with the earlier reports and suggest placental synthesis and differential expression of collectins during spontaneous labor. However, our observations merit further validation with a larger sample size and investigations on the molecular mechanisms regulating the differential expression of collectins at the feto-maternal interface.In conclusion, the study emphasizes that SP-A and SP-D proteins associate with spontaneous labor and plausibly contribute to the anti-inflammatory and pro-inflammatory immune milieu of feto-maternal tissues respectively Fig. 6.Figure S1SP-A, SP-D and MBL transcripts in human term placenta. The real time RT-PCR amplified products were resolved on 2% agarose gel electrophoresis; amplified transcripts SP-A2 (194 bps), SP-D (148 bps), MBL (224 bps) and 18S (171 bps).(TIF)Click here for additional data file.Figure S2Analysis of placental SP-A, SP-D and MBL proteins. A total of 30 µg term placental or 10–30 µg term human amniotic fluid (HAF) proteins were transferred onto the nitrocellulose membrane and probed with monoclonal antibody to a) SP-A or b) SP-D or c) MBL, while negative control was not probed with respective primary antibody (Ab control). The Marker lane shows molecular weight markers in kDa. # non-reducible SP-A dimer (68 kDa); * SP-A monomers in placenta.(TIF)Click here for additional data file.Figure S3Purification of human SP-D from term amniotic fluid. i) Silver stained gel of various fractions, 150 mM MnCl2 eluate (lane 2), 20 mM EDTA eluate (lane 3) and affinity column purified rhSP-D (lane 4); ii) Immunoblotting of various fractions using monoclonal antibody to SP-D, amniotic fluid , term placenta , Mn+2 eluted nSP-D (lane 3) and affinity purified rhSP-D (lane 4).(TIF)Click here for additional data file.Figure S4Cleavage of SP-D oligomers into 25 kDa fragments by MMP9 during spontaneous labor (a) Western blot of term placental tissues using monoclonal antibody to SP-D (n = 3). (b) Densitometric analysis, bars are mean ±SEM, *p<0.05 (student t test). c) MTT assay was performed on the placental explants to test the effect of nSP-D and rhSP-D on their viability. Bars represent mean ±SEM.(TIF)Click here for additional data file.


Helicobacter pylori (H. pylori) infection CD4+ T cells in the gastric lamina propria are hyporesponsive and polarized by Th1/Th17 cell responses controlled by Treg cells. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation, effector function, and induce Treg cells in vitro. In this study, we investigated the underlying mechanisms and the functional relevance of B7-H1 induction by H. pylori infection to chronic infection. Using H. pylori wild type (WT), cag pathogenicity island (cag PAI-) and cagA- isogenic mutant strains we demonstrated that H. pylori requires its type 4 secretion system (T4SS) as well as its effector protein CagA and peptidoglycan (PG) fragments for B7-H1 upregulation on GEC. Our study also showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, which has a functional T4SS delivery system, but not with H. pylori SS1 strain lacking a functional T4SS, led to a strong upregulation of B7-H1 expression in the gastric mucosa, increased bacterial load, induction of Treg cells in the stomach, increased IL-10 in the serum. Interestingly, B7-H1-/- mice showed less Treg cells and reduced bacterial loads after infection. These studies demonstrate how H. pylori T4SS components activate the p38 MAPK pathway, upregulate B7-H1 expression by GEC, and cause Treg cell induction; thus, contribute to establishing a persistent infection characteristic of H. pylori.During  Helicobacter pylori (H. pylori) is a Gram-negative gastroduodenal pathogen. It infects >50% of the world’s population and is linked to chronic gastritis, peptic ulcer disease and gastric cancer (GC) [H. pylori infection usually occurs in childhood and becomes established as a chronic infection. The persistent infection is a major risk factor in the development of GC, the second deadliest cancer worldwide. Overall, H. pylori-associated diseases result in considerable morbidity, mortality and societal costs.cer (GC) –7. H. pyH. pylori, one that is noteworthy is encoded within a 40-kilobase chromosomal region known as the cag pathogenicity island (PAI), which is composed of more than 30 genes that encode for a type 4 secretion system (T4SS). Also, this island of genes includes the cagA gene that codes for the cytotoxin-associated gene A (CagA) protein which is the only known effector protein encoded in cag PAI and is a key virulence factor of H. pylori. Epidemiological studies showed that CagA+H. pylori strains are associated with an increased risk of GC compared to strains of H. pylori lacking CagA [via the H. pylori T4SS [H. pylori peptidoglycan (PG) cell wall fragments into host cells, which are recognized by the intracytoplasmic pattern-recognition receptor (PRR) nucleotide-binding oligomerization domain containing 1 (NOD1). The sensing of H. pylori PG by NOD1 activates NFκB and mitogen-activated protein kinases (MAPKs) and plays an important role in IL-8 production and pathogenesis [Among the multiple virulence factors expressed by ing CagA ,9. The Cori T4SS ,11 and oori T4SS ,13. The ori T4SS ,13, and ori T4SS ,15. In aogenesis –18.H. pylori, it is inadequate to clear the infection. CD4+ Th cells are major effector cells in the immune responses to H. pylori. Although the numbers of CD4+ T cells in the gastric lamina propria with a memory phenotype increase during H. pylori infection, these T cells are hyporesponsive [H. pylori to downregulate T cell responses. One mechanism involves the H. pylori vacuolating toxin A (VacA), which interferes with T cell function by downregulating IL-2 production, IL-2 receptor expression and T cell proliferation [H. pylori also manipulate T cell function by eliciting regulatory T cells (Treg) which are frequently found in H. pylori-infected patients [reg cells further assist in the chronicity of infection. Although H. pylori’s T4SS importance in virulence is recognized because of its multiple effects on GECs, its role in modulating T cell function during H. pylori infection has not been well investigated.Though the host mounts an immune response against sponsive . Becauseferation . H. pylopatients ,22. BecaH. pylori [H. pylori infection and may function locally as APCs; however, their contribution to the response to H. pylori remains understudied. We have previously shown that GECs express cytokines and receptors that influence the T cell responses during H. pylori infection [H. pylori can also use GECs as a fulcrum to inhibit T cell proliferation and cause Treg cell induction from naïve T cells by inducing increased expression of the T cell co-inhibitory molecule B7-H1 on GEC [H. pylori to increase B7-H1 molecule expression on GECs is unknown. In this study we investigated by using in vitro and in vivo systems the role of H. pylori T4SS and two mediators, CagA and PG, translocated into GECs in their increased expression of B7-H1. As both CagA and PG can activate several cell signaling pathways, we also investigated the cell signaling pathways involved in B7-H1 upregulation by H. pylori. Our results showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. Our data also highlighted the in vivo correlation of the presence of functional T4SS delivery system and B7-H1 upregulation with induction of Treg cells in H. pylori infected mice.Professional antigen presenting cells (APCs), such as dendritic cells and macrophages, are important in the regulation of the immune responses against . pylori . GECs arnfection ,25. H. p1 on GEC ,25. B7-Had libitum. The experiments were performed according to an Institutional Animal Care and Use Committee (IACUC) approved protocol (# 0709046A). Feces from sentinel mice housed in the same room were routinely tested by PCR for murine pathogens, including pinworms, mouse parvovirus and consistently tested negative for each of these infections.All mice were kept under pathogen-free conditions, housed in polycarbonate cages on ventilated shelves, with food and water H. pylori strains 51B and 26695 as well as their corresponding isogenic cagA and cag PAI mutants were described previously [H. pylori strains were grown on tryptic soy agar (TSA) plates supplemented with 5% sheep’s blood  or on blood agar plates with 2.5 μg/ml of chloramphenicol  to maintain cagA- [cag PAI- strains at 37°C under microaerophilic conditions. H. pylori strain Sydney strain 1 (SS1) and PM-SS1 (pre-mouse SS1) [Human GECs N87 and AGS were obtained from the American Type Culture Collection (ATCC) and the GEC line HGC-27 was obtained from RIKEN, The Institute of Physical and Chemical Research, Japan. These cell lines were maintained in RPMI 1640 with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. Immortomouse stomach epithelium (ImSt) cells were derived from C57/Bl6 and were maintained in media as described by Whitehead et al. . H. pyloeviously . H. pylon cagA-  and cag use SS1)  were use-/- mice [Helicobacter spp. prior to their use in the experiments. Six-to-eight week old mice were used in the model of gastric H. pylori infection.Female C57BL/6 mice were purchased from the Jackson Laboratory . B7-H1-/- mice  in C57BLPECγ7-conjugated anti-human B7-H1 (clone M1H1), APC-conjugated anti-murine epithelial cell marker EpCAM (clone G8.8) and their isotype controls were purchased from eBioscience. Brilliant violet-conjugated B7-H1 (clone 10F.9G2) and the corresponding isotype control were purchased from Biolegend. The viability dye eFluor 780  was included in the experiments to control cell viability. For cell signaling inhibition the following inhibitors were used: CAY10512 , AG-490 , Wortmannin , and PD169316 . PG-like molecule-NOD1 ligand-iEDAP  was used to investigate the role of PG in B7-H1 expression.H. pylori, GEC were washed and their media replaced with antibiotic-free medium. The bacteria were resuspended in RPMI 1640 medium and used at a cell:bacteria ratio of 1:10.Before infecting the GEC with 6 cells), washed and pre-incubated with normal mouse serum for 15 minute in ice. Cells were washed again and incubated with the corresponding conjugated antibodies or with isotype controls for 30 min in ice. After immunostaining the cells were washed twice with PBS and fixed with paraformaldehyde (1% in PBS). Cells were analyzed by flow cytometry on a LSRII instrument, where at least 104 live events were analyzed on cultured human GEC and 105 cells isolated from murine stomach in order to get 90% interval of confidence. The data were analyzed with BD FACSDiva software  and FlowJo .Flow cytometry was used for surface staining of B7-H1 on cultured GEC lines. Samples were collected after 24-h of incubation with the bacteria or after 12-h, 24-h and 48-h incubation to examine the kinetics of B7-H1 expression after iEDAP treatment. Prior to performing flow cytometry, cells were harvested, counted, their concentration/tube adjusted  and 2 μL of cDNA were added to the PCR reaction step. The reactions were carried out in a final volume of 20 μL using BioRad’s Q5 real-time PCR machine. The cycling parameters were as follows:2 min at 50°C, 10 min at 95°C (1 cycle) and 15 sec 95°C and one min at 60°C (40 cycles).Real-time RT-PCR analysis was performed as previously described . BrieflyTo knockdown NOD1 in GEC, the cells were transfected with siRNA for NOD1 by using the basic nucleofection kit for epithelial cells  according to the manufacturer’s instructions with a cocktail of 0.2 μM of siRNA or a negative control siRNA . For N87 cells, program T-005 was used. Knockdown of expression of NOD1 was verified by real time-RT-PCR.8 CFU (in 100 μL of PBS/inoculation) of H. pylori SS1 or PMSS1 strains, three times within a week. Four weeks later sera were collected as the mice were euthanized and stomachs were removed. Stomachs were dissected longitudinally in 2–4 pieces. Stomach tissue was digested, GECs were isolated and used for immunostaining followed by flow cytometry analysis as we previously described [C57BL/6 mice were orogastrically inoculated with 10escribed .H. pylori infected mice were examined for IL-10 using Luminex array  according to the manufacturer’s instructions. Samples were analyzed using Bio-Plex Manager software (Bio-Rad). After homogenization of mouse stomach tissue, mRNA was isolated and expression of IL-10 and FoxP3 were determined using RT-PCR.Sera collected from H. pylori DNA by real time PCR using a protocol originally described by Rouessel et. al. [H. pylori 16S gene expression and bacterial quantification was performed as described previously [Murine gastric tissue was homogenized and DNA was extracted using DNeasy Blood and Tissue Kit . After purification, the extracted DNA was used for the detection of  et. al. . RT-PCR eviously . For hiseviously .t test for multiple comparisons and considered significant if p was <0.05.Unless otherwise indicated, the results were expressed as the mean ± SE of data obtained from at least three independent experiments done with triplicate sets per each experiment. Differences between means were evaluated by analysis of variance (ANOVA) using student H. pylori-infected and uninfected subjects as well as different human GEC lines that H. pylori infection causes upregulation of B7-H1 expression by GEC. This increased expression of B7-H1 contributes to the inhibition of T cell proliferation and IL-2 production [reg cells in vitro [H. pylori infection. Herein, we sought to determine the underlying mechanisms leading to B7-H1 upregulation by H. pylori. As the H. pylori cag PAI encoded T4SS is important in delivering bacterial products  that alter multiple properties of the gastric epithelium, we hypothesized that this virulence factor could influence B7-H1 upregulation. To test this hypothesis, we infected the human GEC lines  with H. pylori 51B wild type (WT) strain which has an intact cag PAI and with an isogenic mutant strain lacking the cag PAI, H. pylori 51B cag PAI-. Our flow cytometry results showed that while the H. pylori WT strain caused a significant upregulation of B7-H1 expression in GECs, the mutant strain lacking cag PAI failed to upregulate B7-H1 expression (cag PAI-dependent upregulation of B7-H1 expression by H. pylori. Similar data were obtained with a different set of WT and cag PAI- strains (H. pylori 26695). All experiments in vitro were performed with the three human GEC lines listed earlier to confirm that the results were consistent and not cell line-dependent.We previously showed using gastric biopsy samples from oduction . Interesin vitro . These rpression , which sH. pylori uses its T4SS to translocate into GECs the effector protein CagA and cell wall PG fragments [H. pylori mediated upregulation of B7-H1 depends on the presence of T4SS. To dissect the role of T4SS components in B7-H1 upregulation we investigated the role of T4SS translocated CagA and PG fragments on B7-H1 increased expression. To examine whether CagA plays a role in B7-H1 upregulation we infected human AGS cells with H. pylori 51B cagA+ (WT) and a cagA- strain and assessed B7-H1 mRNA expression using real time RT-PCR. H. pylori WT strain over the levels for untreated controls after 2-h of infection. In contrast, the H. pylori cagA- strain did not affect B7-H1 mRNA expression. Our flow cytometry data also showed that H. pylori 51B cagA+ strains induced significant upregulation of B7-H1 expression on the surface of AGS cells compared to AGS infected with a cagA- mutant strain  stimulated with the synthetic PG analogue, iEDAP, that is a NOD1 ligand, had more than two fold upregulation of B7-H1 mRNA expression within two hours of incubation  were treated with ten-fold different concentrations of iEDAP and B7-H1 expression was analyzed by flow cytometry. The lowest concentration of iEDAP which elicited a significant upregulation of B7-H1 was 10 μg/ml, but it did not change much by increasing it to 100 μg/ml iEDAP as the response was similar to that with 10 μg/ml iEDAP . SimilarH. pylori and other gram negative bacteria are recognized by the intracellular NOD1 receptors and cause activation of NFκB and MAPK pathways [H. pylori mediators that could influence the modulation of B7-H1 expression. To that end, we used different pharmacological inhibitors directed against NFκB (CAY10512), p38 MAPK (PD169316), STAT3 (AG-490) and PI3K (wortmannin) pathways, which are known to be activated by H. pylori [H. pylori  we showed that infection with this strain causes a significant increase of B7-H1 expression in murine GECs and the response is dose dependent  are present in higher numbers in the gastric mucosa of H. pylori-infected persons and play an important role in regulating the inflammatory response and inhibiting bacterial clearance [H. pylori upregulates the expression of the T cell co-inhibitory molecule B7-H1 by human GEC, which in turn inhibit T cell proliferation [reg cells [H. pylori upregulate B7-H1 were unknown and could represent important targets in vaccine design. Using human GEC in an in vitro model complemented with an in vivo mouse model of infection which included both WT and B7-H1-/- mice, we showed that H. pylori uses T4SS to translocate the effector protein CagA and PG cell wall fragments to upregulate B7-H1 expression by GECs. This increased expression of B7-H1 by GEC promotes the expansion of Treg cells which may foster bacterial persistence. Our study also explored the underlying cell signaling pathways involved in this mechanism and showed that H. pylori uses the p38 MAPK pathway for the upregulation of B7-H1 expression.esponses –46. Howelearance ,47. Prevferation  and causH. pylori upregulates B7-H1 in GEC [Porphyromonous gingivalis in oral squamous carcinoma cells and also hepatitis B virus-infected myeloid dendritic cells have been reported to increase B7-H1 [H. pylori T4SS has been shown to play an important role in inflammation, pathogenesis and immune evasion mechanisms during H. pylori infection [H. pylori WT, cag PAI- and cagA- mutant strains we showed that the upregulation of B7-H1 by H. pylori depends on the presence of T4SS and its effector protein CagA. Though in our initial studies we did not reveal a significant difference in B7-H1 expression levels in GEC infected with H. pylori WT and cag PAI mutant strain, this time using several GECs and infecting them with H. pylori clinical strain and its corresponding mutant we showed that H. pylori cag PAI and its effector protein CagA play an important role in upregulating B7-H1 at both the mRNA and protein levels. The previous attempt to determine the role of H. pylori T4SS in B7-H1 upregulation may not have revealed differences between wild type and cagA mutants in B7-H1 induction due to differences in the growth kinetics of the WT and mutant strains, which had not been studied. This time we studied the growth kinetics of the mutants and the corresponding parental strains and infected the GEC with the same CFU of the mutant and parental strains.B7-H1 (PD-L1) is a member of the B7 family which interacts with its putative receptor PD-1 and plays an important role in cell-mediated immune responses ,49. B7-H1 in GEC . Subsequse B7-H1 ,53. Sincnfection , we inveH. pylori-mediated B7-H1 upregulation was only partially dependent on CagA injection. This suggested that other components of T4SS are also involved in this mechanism. Previous studies highlighted the importance of H. pylori T4SS secreted component PG in activation of MAPK and NFκB pathway and induction of inflammation [H. pylori uses its HP310 protein for PG N-deacetylation which contribute to H. pylori’s survival in the host [H. pylori PG in B7-H1 expression. Our flow cytometry and RT-PCR data clearly showed that H. pylori PG plays an important role in the upregulation of B7-H1 in GEC. By silencing NOD1 expression in GEC using siRNA nucleofection, we confirmed that recognition of PG by NOD1 contributes to this modulation of B7-H1 by H. pylori. To our knowledge, this is the first report showing the ability of H. pylori PG in modulating the immunoregulatory properties of GEC, specifically in contributing to inhibition of host T cell function.Our data also showed that ammation . Though ammation , the actammation . A recenthe host . In thisH. pylori mediated activation of host cell signaling pathways plays an important role in changing the homeostasis of GEC, which is very important for regulation of local T cell responses, we also determined the cell signaling pathways used by H. pylori to modulate B7-H1 expression in GEC. We focused on the cell signaling pathways that are known to be activated by H. pylori T4SS, e.g. NFκB, STAT3, MAPK and PI3K pathways [H. pylori activates STAT3 to modulate host immune responses [H. pylori CagA was reported to increase the bacterial lectin regenerating islet-derived (REG)3γ expression in GECs via activation of the STAT3 pathway, which allows H. pylori to manipulate host immunity to favor its own survival in the gastric environment [H. pylori IL-8 production [H. pylori T4SS manipulates host immune regulation and deregulates gastric epithelial homeostasis for their survival. Using cell signaling inhibitors we showed that H. pylori uses p38 MAPK pathway to upregulate B7-H1 expression in GEC, since treating cells with the MAPK p38 inhibitor PD169316 prior to infecting the GEC with H. pylori inhibited B7-H1 induction by the pathogen. Thus our study showed another novel mechanism where H. pylori uses host cell signaling pathways to change the properties of GEC and thus makes a safer environment for bacterial survival.As pathways ,35,39–42esponses ,36. A reoduction . MAPKs aoduction . These oin vivo using a mouse model of infection. Infection with the H. pylori PMSS1 strain, which contains a functional T4SS and can deliver CagA and PG into GEC, showed upregulation of B7-H1 molecule in both murine GEC line (ImSt) and also in the mouse model. However, H. pylori SS1 strain which lacks this delivery system failed to upregulate B7-H1 in both ImSt murine epithelial cells and in GEC isolated from infected murine gastric mucosa. Taken together our in vitro and in vivo data confirmed that H. pylori uses its T4SS component CagA and PG to increase T cell–coinhibitory molecule B7-H1 on GEC which plays an important role in controlling T cell activation.The role of T4SS was further confirmed in a murine GEC line and H. pylori infected patients have increased numbers of Treg cells (CD4+ CD25high FoxP3+) in their gastric mucosa [reg cells are important for suppressing overall T cell response during infection and cancer, they also inhibit protective effector T cell activity by producing anti-inflammatory cytokines IL-10 and TGF-β [reg cells also inhibit memory T cell response in the periphery [reg cells from naïve CD4+ T cells in a B7-H1 dependent manner. Tregs cause inhibition of T cell proliferation and this process depends on B7-H1 [reg cell development from naïve CD4+ T cells [H. pylori-infected GEC with naïve T cells our lab showed that H. pylori-mediated induction of B7-H1 co-related with induction of Treg cell development from naïve T cells [in vivo and also determined the bacterial virulence factor involved in this mechanism. Our study showed that mice infected with H. pylori PMSS1 strain have increased levels of circulating IL-10, an important mediator of Treg cell function and increased Treg cell transcription factor FoxP3 mRNA expression in their stomachs after four weeks of infection. In contrast, infection with SS1 strain, which lacks a functional T4SS, failed to upregulate IL-10 production and FoxP3 expression. IL-10 produced by Treg cells is critical for their function as FoxP3+ T cells lacking IL-10 are unable to suppress the development of gastritis and colitis [-/- mice we showed that H. pylori uses B7-H1 to foster Treg cell development since the B7-H1-/- mice infected with H. pylori had reduced Treg cell numbers compared to the WT mice. Although we observed a trend in the increase on inflammatory cells within the gastric mucosa of H. pylori-infected B7-H1-/- mice compared to similarly infected wild type mice, it did not reach statistical significance at the time that we examined. Further investigation is needed to determine whether differences in inflammation are more pronounced at other time points. A previous study showed that Treg cells play a protective role against gastric inflammation by reducing inflammation and ulceration in children [reg cell contribute to the chronicity of H. pylori infection. Work by Rad et al. [reg cells have dual function in inhibiting gastric inflammation and facilitating bacterial colonization. Though inhibition of bacterial-induced inflammation is important to reduce the extent of gastritis and ulcer formation, it is also important to clear the bacteria from the system to inhibit chronic infection. This study further supports our previous findings in vitro and showed that H. pylori mediated modulation of B7-H1 expression in GEC causes an increased Treg type response and validate the hypothesis that Treg induction aids the bacteria to survive in the gastric mucosa and further demonstrated that the pathogen is using its T4SS components for this mechanism. Taken together our studies showed for the first time how H. pylori use GEC as mediators to manipulate local Treg cell response by using its T4SS components to aid in H. pylori persistence. These findings have significant importance as they provide insights into the H. pylori virulence factor involved in inhibiting host immune response and the underlying cell signaling pathway involved in this mechanism. Both the virulence factors and cell signaling pathways involved are potential targets to design a therapeutic or a vaccine to circumvent this highly prevalent human pathogen.c mucosa . Though nd TGF-β ,58. Tregeriphery . A previon B7-H1 . Francis T cells . Previou colitis . In addichildren . In contchildren  suggested et al.  showed tS1 FigH. pylori:GEC ratio for 24-h and B7-H1 expression was measured by immunostaining followed by flow cytometry. The data expressed as mean fluorescent intensity. Isotype control value was subtracted from the data presented. N = 8,*P < 0.05, ** P < 0.01 and *** P < 0.001.N87 cells were infected with PMSS1 strain  or SS1 strain  at 10:1 (TIF)Click here for additional data file.


In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A–D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin.  Prosaposin (PS) is the precursor of saposins A–D and is predominantly expressed in the brain, muscles PS has two isoforms, Pro+9 and Pro+0, caused by differential splicing at the saposin B domain In general, neurons stop proliferating in adulthood. In the hippocampal dentate gyrus and the olfactory bulb in adult mammals, however, neurogenesis lasts throughout life Neuronal precursor cells are distributed throughout the hilus of the dentate gyrus in the early stages after birth but gradually locate to the subgranular zone (SGZ) facing the hilus where they differentiate as they grow in situ hybridization assays of Pro+0 and Pro+9 to investigate PS production in the dentate gyrus.The dentate gyrus in the hippocampus, which plays a crucial role in memory formation, is one of two brain regions in which neurogenesis occurs even in adulthood The protocol for this trial and supporting ARRIVE checklist are available as supporting information; see g. The heat-stable fraction was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes. After blocking, the membrane was immunolabeled with rabbit anti-saposin B, D, and PS. Immunoreactions were visualized using horseradish peroxidase-conjugated anti-rabbit immunoglobulin and enhanced chemiluminescence . Immunoreactive protein bands were visualized with an LAS-4000 luminescence image analyzer . The specificity of the anti-saposin B and D antiserum has been previously reported using Western blotting A specific polyclonal antibody against PS was generated by immunizing rabbits with a synthetic oligopeptide corresponding to the proteolytic portion of rat PS  (409-PKEPAPPKQPEEPKQSALRAHVPPQK-434) Male Wistar rats aged P3, P7, P14, and P28 were used in group of six. All animals were housed at a constant temperature (22°C) under a 12∶12-h light–dark cycle and given food and water ad libitum. The following experiments were conducted in accordance with the Guide for Animal Experimentation at Ehime University School of Medicine, Japan. The current study, in addition to its protocol, was approved by the Committee on the Ethics of Animal Experiments of the Ehime University School of Medicine (Permit Number: A21-2). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. After anesthetizing by intraperitoneal injection of chloral hydrate (10 mg/kg), animals were transcardially perfused with heparinized phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer . Brains were removed and post-fixed overnight in the same fixative. They were immersed in 0.1 M PB containing 30% sucrose and frozen on dry ice. Coronary sections (5 µm thick) were cut using a cryostat, thaw-mounted onto silane-coated slides, and stored at −80°C until use.The anti-PS antibody was used to evaluate PS. As noted above, this antibody recognizes the intermediate sequence between saposin C and saposin D, and does not react with saposin A, B, C, or D. Its use as a specific antibody was demonstrated by immunoblotting. NeuN and PSA-NCAM were used as markers to distinguish immature or mature granule cells in the dentate gyrus. NeuN is globally used as a marker of mature neurons that are past cell division, whereas PSA-NCAM is useful as a marker of relatively immature neurons with axons and dendrites in the postmitotic differentiation step. Localizations of PS, NeuN, and PSA-NCAM were examined using these antibodies, and the relationship to neurogenesis was evaluated.3, followed by incubation with the primary antibodies: rabbit polyclonal anti-PS IgG (1∶250), mouse monoclonal anti-PSA-NCAM IgM (1∶1000), or mouse monoclonal anti-NeuN IgG (1∶1000). Sections were washed twice for 15 min in PBS and reacted with the secondary antibodies [Cy3 anti-rabbit IgG (1∶500) and FITC anti-mouse IgG and IgM (1∶300)]. DAPI was added for 2 h to counterstain the nuclei. Finally, sections were washed twice for 15 min in PBS, mounted using Permafluor Aqueous Mounting media , and observed using fluorescence microscopy .Cryosections were air-dried and washed for 15 min in PBS plus Tween 20 (PBS-T). Sections were blocked overnight in PBS (0.1 M) containing 5% bovine serum albumin, 5% normal swine serum, and 0.1% NaNLaser microscopy was used to visualize the cellular localization patterns of PS using immunofluorescence. Sections (1-µm thickness) from the dentate gyrus of postnatal day (P) 28 rats were triple-stained with antibodies against polysialylated-neural cell adhesion (PSA-NCAM), saposin B and 4′,6-diamidino-2-phenylindole (DAPI) and observed using laser microscopy. Additionally, 1-µm sections from rats at P1, P7, P14 and P21 were triple-stained with antibodies against PS, microtubule-associated protein (Map-II) and DAPI, and assessed using laser microscopy. High-resolution confocal images were taken using an A1 confocal microscope equipped with a 60× oil immersion lens . Single-plane 1024×1024 images were captured using a 33.4 µm pinhole with 3× zoom.in situ hybridization. Three antisense 36-mer oligonucleotide probes AS1, AS3, and AS4, and one sense probe S1, were commercially synthesized . AS1 was complementary to bases 1704–1739 in the 3′-untranslated region of the PS cDNA, AS3 contained the 9-base insertion to detect Pro+9 mRNA, and AS4 excluded the 9-base insertion and so it detected Pro+0 mRNA. For the control, the sense probe S1 was complementary to AS1. The sequences of the four probes are as follows:Brains were removed, frozen on dry ice, and cut into 10-µm sections using a cryostat for 5′- TTCATTACCCTAGACCCACAAGTAGGCGACTTCTGC-3′•AS1: 5′- GCAGAAGTCGCCTACTTGTGGGTCTAGGGTAATGAA-3′•S1: CTTGGGTTGCTGATCCTGCATGTGCATCATCATCTG-3′5′-•AS3: TTCCTTGGGTTGCATGTGCATCATCATCTGGACGGC-3′5′-•AS4: 35S-labeled antisense or sense probes (1.0×107 cpm/ml) in hybridization buffer . After hybridization, sections were rinsed three times with 1× SSC at 55°C for 20 min, dehydrated through a graded ethanol series, coated with NBT2 emulsion , and exposed for 3 weeks at 4°C. Finally, sections were developed using D-19 developer (Eastman Kodak) and observed under a dark-field microscope.Frozen sections were fixed for 15 min in 4% paraformaldehyde in 0.1 M PBS (pH 7.4), rinsed with standard 4× saline citrate , and dehydrated through a graded ethanol series. Sections were hybridized overnight at 41°C with in situ hybridization without hematoxylin and eosin (HE) counterstaining. The average gray value of all pixels in each image was determined using the NIH 1.56 software (public domain software by Dr. Steve Barrett). The statistical significance of the values was examined by one-way analysis of variance (ANOVA) and Scheffe's test using the StatView software . P-values <0.05 were considered to indicate statistical significance.We obtained digital images of the PS hybridization signals in the dentate gyrus sections, as well as images after To examine the specificity of the rabbit anti-PS antibody, its reactivity was compared with the specificity of rabbit anti-saposin B and D antibodies using immunoblotting. Two bands were detected upon immunoblotting with the anti-saposin B and D antibodies a,b. TheOur immunoblotting results show that the rabbit anti-PS antibody used in this study specifically recognizes PS and does not recognize di- or trisaposin. Therefore, the specificity of the anti-PS antibody was demonstrated.In neurons from the dentate gyrus at P3 and P7, we observed stronger PS immunoreactivity at P14 a–d . ImPSA-NCAM immunoreactivity was clearly observed in the cytoplasm and dendrites e–h. TheBased on the localization of PS-single positive cells in the outer granule cell layer d and PSA few double-positive cells were observed at P7 j  and sThe results of prosaposin immunofluorescence a–d  werDouble-positive cells were widely observed in all sections. Only DAPI-positive, PS-, and NeuN-double negative cells were observed in the SGZ i–l . A Saposin B immunoreactivity is frequently detected in PSA-NCAM-positive cells in the SGZ where PS immunoreactivity is rarely observed a,b. TheLaser microscopy images at higher magnification  were exaa–l ). As comparison of image analyses among the stages was difficult, we compared the hybridization signal intensities among the sections in the same stage treated with different probes. All sections without HE counterstaining (m–u) showed greater expression of Pro+9 mRNA than Pro+0 mRNA (v–x). Higher-magnification images with HE counterstaining revealed that neurons in the subgranular zone (SGZ) showed weak Pro+9 expression at P21, in agreement with PS immunoreactivity. The highest Pro+9 mRNA expression was observed in CA4 neurons in the hilus, and moderate signals were observed in a number of interneurons in the molecular layer  or against PS and NeuN (a marker of mature neurons).Furthermore, we analyzed the expression of the secretory type (Pro+9) or lysosomal type (Pro+0) of PS mRNA in the developing dentate gyrus from P3 to P21., To illustrate our results, the development of PS-positive neurons at each stage is summarized in a scheme shown in Neuronal differentiation in the dentate gyrus was not complete at P7, and PS was synthesized in immature granule cells at this time b, 3b. PSA-NCAM-positive cells predominantly exist in the SGZ, the basal side of the granule cell layer of the dentate gyrus, but PS-positive cells localize to the apical side of the granule cell layer at P28. This study shows that most PS-positive cells in the dentate gyrus at P28 are NeuN-positive mature neurons. Double-positive neurons were rarely observed in the SGZ at P28. PSA-NCAM-positive cells in the SGZ extend their dendrites through the granule cell layer composed of many PS-positive neurons Click here for additional data file.


Overexpressed CEACAM6 induced epithelial-mesenchymal transition (EMT) in GC, as measured by increases in the EMT markers N-cadherin, Vimentin and Slug while E-cadherin expression was decreased in CEACAM6-overexpressing GC cells; opposing results were observed in CEACAM6-silenced cells. Furthermore, E-cadherin expression was negatively correlated with depth of tumor invasion, lymph node metastasis and TNM stage in GC tissues. Additionally, CEACAM6 elevated matrix metalloproteinase-9 (MMP-9) activity in GC, and anti-MMP-9 antibody could reverse the increasing invasion and migration induced by CEACAM6. CEACAM6 also increased the levels of phosphorylated AKT, which is involved in the progression of a variety of human tumors. We further observed that LY294002, a PI3K inhibitor, could reverse CEACAM6-induced EMT via mesenchymal-epithelial transition. These findings suggest that CEACAM6 enhances invasion and metastasis in GC by promoting EMT via the PI3K/AKT signaling pathway.Overexpressed CEACAM6 in tumor tissues plays important roles in invasion, metastasis and anoikis resistance in a variety of human cancers. We recently reported that CEACAM6 expression is upregulated in Gastric cancer (GC) tissues and promoted GC metastasis. Here, we report that CEACAM6 promotes peritoneal metastases  GC is one of the most common malignant tumors and a major health issue worldwide, particularly in East Asian countries such as Japan, Korea, China, where it is the second cause of cancer-related death Epithelial-mesenchymal transition (EMT) is not only a physiological process during embryonic development or tissue regeneration, but also a pathological element in cancer progression, involving tumor metastasis, apoptosis and senescence resistance In this study, we further examined the effects and potential pathways of CEACAM6 in GC invasion and metastasis, and investigated its correlation with EMT.Written informed consent in the study has been obtained from all participants. The study protocol was approved by the ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Animal procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Jiao Tong University, Shanghai, China.2 and saturation humidity in RPMI-1640 medium containing 10% fetal bovine serum.The human GC cell lines SGC-7901, MKN-45 and MKN-28 were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured at 37°C in 5% COGastric tumor and adjacent non-tumorous tissue were obtained from 93 patients with GC who underwent curative surgery at Shanghai Jiaotong University School of Medicine Affiliated Ruijin Hospital from 2010 to 2013. The patients consisted of 69 men and 24 women with a mean age of 62.1 years . None of the patients had received radiotherapy or chemotherapy prior to surgery. Clinicopathological data were collected and pathological tumor staging was determined according to the UICC TNM classification. Histological typing was performed by at least two expert pathologists working independently in a double-blinded fashion. This study was approved by the Ethics Committee of Shanghai Ruijin Hospital, and all patients were fully informed of the experimental procedures.5′-CCGGAATTCCCATGGGACCCCCCTCAGCCC-3′ (forward) and 5′-TCCCCCGGGGCTATATCAGAGCCACCCTGG-3′ (reverse). We assembled a pIRES2-eGFP-CEACAM6 construct by inserting CEACAM6 cDNA into pIRES2-eGFP vector. We next transfected pIRES2-eGFP-CEACAM6 or pIRES2-eGFP vector into SGC-7901 and MKN-45 cells using Lipofectamine 2000  in accordance with the manufacturer’s protocol. Stable clones were selected by continuous treatment with G418 .Full-length CEACAM6 cDNA was obtained by RT-PCR from total RNA extracted from GC samples. The primer sequences were 5′-CACCGCCGGACAGTTCCATGTATACGAATATACATGGAACTGTCCGG-3′ (forward), and 5′-AAAACCGGACAGTTCCATGTATATTCGTATACATGGAACTGTCCGGC-3′ (reverse); control, 5′-CACCGCTACACAAATCAGCGATTTCGAAAAATCGCTGATTTGTGTAG-3′ (forward), and 5′-AAAACTACACAAATCAGCGATTTTTCGAAATCGCTGATTTGTGTAGC-3′ (reverse). CEACAM6 shRNA was subcloned into pL/shRNA/F lentiviral vector to obtain a pL/shRNA/shR-CEACAM6 construct. Then pL/shRNA/shR-CEACAM6 or control lentiviral vectors were transfected into MKN-28 GC cells. Stably transfected cells were selected by treatment with 5 µg/ml blasticidin and were used for identification and further research.Based on human CEACAM6 gene data, a pair of oligonucleotide sequences and negative control sequences were designed and synthesized by Shanghai Novobio Scientific Co., Ltd. shRNA sequences were as follows: CEACAM6, Cells were harvested and lysed using RIPA buffer  containing 1% PMSF protease inhibitors. A BCA assay kit  was used to measure the total protein concentration. Equivalent amounts of protein were separated by 10% SDS-PAGE, and the resolved proteins transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 2 h and then incubated with primary antibodies overnight at 4°C. Primary antibodies were as follows: CEACAM6 , E-cadherin , USA), N-cadherin , Vimentin , Slug , p-AKT (Ser473) , total AKT  and GAPDH . Membranes were then incubated with secondary antibody for 2 h at room temperature and were visualized using an enhanced chemiluminescence detection system  in accordance with the manufacturer’s protocol.Cells were cultured on coverslips for 24 h and then fixed with 4% paraformaldehyde for 15 min. Cells were washed with PBS, then permeabilized with 0.5% Triton X-100 for 20 min and blocked with 5% BSA for 30 min at room temperature. We next stained the cells with CEACAM6 antibody  at 37°C for 2 h, followed by incubation with fluorescent secondary antibody for 1 h at room temperature. The nuclei were stained with DAPI. Rhodamine phalloidin antibody  was used to visualize the cytoskeleton of GC cells. Slides were analyzed and imaged on a fluorescence microscope.Sections of 4 µm-thick were cut from paraffin-embedded tissue blocks and then deparaffinized and rehydrated. Immunohistochemical staining of sections was performed according to the DAKO protocol, using mouse anti-CEACAM6  and E-cadherin  at 4°C overnight. Slides were then incubated with HRP-labeled secondary antibody and visualized by diaminobenzidine. Two pathologists who were blinded to any patient data independently evaluated and scored the sections. Immunohistochemistry stain score = positive cell score + staining intensity score. The percentage of positive cells was scored as follows: 0 (10%), 1 (10–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). Immunohistochemical staining intensity was graded as follows: 0 (no staining), 1 (weak staining), 2 (brown staining), and 3 (dark brown staining). Total scores of ≥3 were defined as positive to simplify data analysis. To analyze the correlation between CEACAM6 and E-cadherin expression, Image-Pro Plus version 6.0  was used to quantify the expression of CEACAM6 and E-cadherin on 20 samples.To examine MMP-9 activity, zymography was performed using 10% SDS-PAGE gels containing 1 mg/ml gelatin (Sigma). Briefly, GC cells were cultured in serum-free RPMI-1640 medium for 24 h and the supernatant was then collected and centrifuged at 1000 rpm for 5 min. The gels were washed twice in renaturation buffer (2.5% Triton X-100) for 30 min each time to remove SDS after electrophoresis and then incubated at 37°C for 24 h in a reaction buffer . We next stained the gels with 0.5% Coomassie brilliant blue R-250 for 2 h and destained the gels in buffer . Clear transparent bands in the background of blue staining represented the gelatinase activities.5 cells were suspended in serum-free RPMI-1640 with or without MMP-9 antibody  and plated in transwell chambers  with or without Matrigel  according to the manufacturer’s protocols. For both assays, medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h culture, cells were fixed by 10% formalin and stained by 0.5% crystal violet. Finally, cells in the lower chamber were photographed and counted by inverted microscopy.For cell migration and invasion assays, a total of 1×10invivo. Nude mice were housed at a specific pathogen-free environment in the Animal Laboratory Unit, School of Medicine, Shanghai Jiao Tong University, China. Mice received humane care and the study protocols were carried out according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Jiao Tong University, Shanghai, China. A total of 2×106 SGC-7901-CEACAM6 or SGC-7901-NC cells were injected into five mice abdominal cavity of each group (randomisation). Mice were euthanized on the 30th day after injection, and the abdominal masses were imaged and photographed. All experiments were performed in accordance with the official recommendations of the Chinese animal community.Four-week-old male BALB/c nude mice  were used to evaluate the role of CEACAM6 in peritoneal spreading t-test was used to examine the statistical differences between the two groups. Correlations between E-cadherin expression in GC tissues and clinicopathological parameters and CEACAM6 expression were analyzed by chi-square or Fisher’s exact tests or Pearson correlation coefficient analysis. Data are shown as mean±SD. P<0.05 and P<0.01 was considered statistically significant and highly significant, respectively.Student’s Our previous results implied that MKN-28 GC cells inherently express high levels of CEACAM6, whereas SGC-7901 and MKN-45 GC cells express low levels Interestingly, cell morphology was altered following CEACAM6 attenuation and overexpression. Compared with the control cells, SGC-7901-CEACAM6 and MKN-45-CEACAM6 cells exhibited a more mesenchymal-like morphology, and were spindle- and fusiform-shaped . ConversP = 0.046), lymph node metastasis (P = 0.013), and TNM stage (P = 0.035), but not with other clinicopathological factors including sex, age, or differentiation . CEACAM6 expression in tumor tissues was higher than that in matched non-tumorous tissues , consistEMT-related proteins in GC cells were evaluated by western blot analysis. We observed that the N-cadherin, Vimentin and Slug protein levels were increased, while E-cadherin was decreased in CEACAM6-overexpressing cells compared with their respective control cells . ConversP<0.01; It is well known that adhesion, degradation and migration are major issues in cancer metastasis. Gelatin zymography was carried out to examine the effects of CEACAM6 on the activity of MMP-9, which is important in ECM degradation. MMP-9 activity in SGC-7901-CEACAM6 and MKN-45-CEACAM6 cells was increased compared with their control groups . We nextWe previously observed that SRC activity was induced by CEACAM6 in SGC-7901 GC cells. AKT is a downstream protein of SRC and is dramatically associated with EMT, invasion and metastasis in tumor progression. We therefore evaluated AKT activity in CEACAM6-overexpressing GC cells. Western blotting showed that AKT phosphorylation at serine 473 was markedly increased in CEACAM6-overexpressing GC cells compared with the control groups . To examinvivo. We injected SGC-7901-CEACAM6 and SGC-7901-NC cells into the abdomens of nude mice. Extensive peritoneal spread was observed in the SGC-7901-CEACAM6 group compared with the SGC-7901-NC group  in 98 GC tissues in our previous study Interestingly, we observed that cell morphology and cytoskeletal structure was altered by stable upregulation and downregulation CEACAM6 in GC cells. CEACAM6-overexpressing cells were more mesenchymal-like and exhibited a spindle and fusiform shape, and more actin stress fibers were detected compared with the control groups, in a process defined as EMT. The mesenchymal phenotype endows cells with more migratory and invasive properties Metastasis is the leading cause of cancer-associated death but has been difficult to study because it involves a series of rare, stochastic events. A number of potential signaling pathways relevant to cancer metastasis have been illustrated, including the PI3K/AKT, MAPK/ERK, FAK-SRC, JAK/STAT, NF-kβ, and MMPs pathways invitro and invivo, and may be a potential new diagnostic and prognostic marker and novel target for GC therapy.In conclusion, we have illustrated that CEACAM6 acts as an oncogene by promoting EMT via PI3K/AKT activation in GC. Furthermore, E-cadherin is markedly downregulated in GC tissues than paired adjacent non-tumorous tissues. CEACAM6 promotes invasion and metastasis Checklist S1The ARRIVE guidelines. All in vivo experiments in our manuscript were exported in accordance with the ARRIVE guidelines.(DOC)Click here for additional data file.


Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of  TFs can be involved in transcriptional activation, repression or both. TFs possess a modular structure generally comprising a DNA-binding domain (DBD) together with a regulatory or sensing domain. Specific signatures present at the amino acid level (notably into the DBD) have allowed categorizing TFs into various families NGTT[G/A]  and the AC-elements that are AC-rich sequences , which recognise the [A/T/G][A/T/G]C[C/G][C/G] consensus sequence [Malus domestica) MdMYB10 that interacts with the ACTGGTAGCTATT DNA motif [R2R3-MYBs control the expression of their target genes through the interaction with specific DNA sequences usually present upstream of the transcribed region within the promoters . These c[A/C/T]; , 14). Unsequence , or the NA motif , 16.e.g. ) sequences are specific to R2R3-MYB proteins belonging to clade A  whereas clade B R2R3-MYBs (i.e. subgroup 18) interacted equally with either type I and type II (TNGTT[G/A]) MYB-core sequences. All the other R2R3-MYBs (clade C) were considered as more specific to AC-rich sequences (ACC[A/T]A[A/C][T/C]). However, a substantial amount of data gathered from various studies revealed that this classification was not fully accurate  using the Gateway® technology with and without the stop codon in order to keep the field of application of this library as broad as possible . Binding activities were assayed in vivo in Y1H experiments with a pool of 16 DNA sequences. These DNA motifs are well-characterised cis-regulatory elements, known to interact with R2R3-MYB proteins, that belong to both, the MYB-core and AC-rich groups CCAAC) at their junction, which is similar to well described AC-II element  associations between a specific DNA motif from group II . Similaequences , 30. In in planta by numerous R2R3-MYB has been identified ) cis-regulatory sequences ([A/T/G][A/T/G]C[C/G][C/G] consensus sequence ) was the most represented, and that the GTT[A/G] motif was strongly conserved for all DBDs. No LOGO was generated for S24 DBD, reflecting its low BS for this group of DNA sequence  were associated with a LOGO that corresponded to a unique DNA motif because of their overall low BS , which is similar to the type II MYB-core consensus motif (TNGTT[A/G]). Three DBD subgroups associated with a LOGO corresponding to a unique AC-rich motif, namely S19, S24 and SAt46. If this finding was expected for S24 and SAt46, the absence of a similar result for S1, S2, S3, S13 and S24 DBDs was surprising . This ictively; ) as wellR3-MYBs; ).i.e. representative of the R2R3-MYB and/or DBD subgroups) would undoubtedly be ideal in order to enhance our ability to understand and predict the interaction occurring between the R2R3-MYBs (or DBDs) and their DNA targets. Nevertheless, if this type of approach could generate crucial information notably on the dynamic and tissue specificity of these interactions, its set up remains technically demanding (when feasible), time consuming and its cost relatively expensive. An alternative would be to pursue the Y1H approach by extending the interaction matrix by increasing the number of assayed DNA motifs, ideally in a quantitative manner (e.g. Y1H experiments using LacZ as reporter gene), and to include the R2R3-MYBs that are specific to woody species . Independently of the method used, this later point would allow determining if some DNA binding specificities have arose in perennial woody species NGTT[A/G]. (B) Logo generated from the 21 DNA motifs. (C) Logo generated from the DNA motifs that were similar to the MYB-core type I sequence: CNGTT[A/G]. (D) Logo generated from the DNA motifs that were similar to the MYB-core type II sequence: TNGTT[A/G].(PDF)Click here for additional data file.S6 FigA) Transient expression assays: green fluorescent protein (GFP) intensity was measured in Physcomitrella patens protoplasts co-transfected with proBAN76:35Smini:GFP alone (negative control) or together with AtTT8, AtTTG1 and AtTT2. TT2 is a key transcriptional regulator of proanthocyanidin (PA) biosynthesis (flavonoid) in seeds. TT2, together with TT8/bHLH042 and TTG1  form a ternary protein complex that specifically regulates the expression of genes involved in this pathway, such as BANYULS. The origin of AtTT2 being from either a previous study  Arabidopsis thaliana mutant complementation experiments: (B) myb5 tt2 transparent testa phenotype (i.e. yellow seeds deprived of PAs) was complemented by expressing TT2/AtMYB123 or AtMYB5 under the control of the TT8 promoter as previously described [C) gl1-1 lack of trichomes was reverted by overexpressing GL1 (GLABRA1/AtMYB0) and WER (WEREWOLF/AtMYB66) ORFs, encoding functional homologues. that coescribed , (C) gl1(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950T and Mycobacterium yongonense DSM 45126T. Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126T than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes  and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum.Recently, we introduced a distinct  Mycobacterium avium complex (MAC) are the most important nontuberculous mycobacteria (NTM) in terms of clinical and epidemiological aspects   , 17 were_021715) , M. para016947)] –24. Thes016947)]  by the MM. intracellulare strains , 2 INT-5 strains (MOTT-H4Y and MOTT-36Y) and M. yongonense DSM 45126T, the rpoB gene and 35 additional gene sequences were selected from the genome sequences. In the selected 35 genes, 10 genes  -superoxide dismutase (sodA)   were ince (sodA) . The retsoftware . Using tsoftware  in the Msoftware . The boorpoB gene sequences and the 35 selected gene sequences from the 3 M. intracellulare strains , 2 M. intracellulare INT-5 strains (MOTT-H4Y and MOTT-36Y), M. yongonense DSM 45126T and M. parascrofulaceum ATCC BAA-614T. Then, the patterns were compared. Additionally, the extracted SNP sequences were concatenated and used to construct a phylogenetic tree as described above.SNPs were extracted from the multiple alignments of M. intracellulare strains (INT-1 or INT-2 strains), five genes of other M. intracellulare clinical isolates, which proved to have more M. yongonense-group related signature SNPs than others, were amplified and sequenced for further analysis. The five selected genes were argH, dnaA, deaD, hsp65 and recF. Genomic DNA from each of the five INT-1 strains , five INT-2 strains , and four INT-5 strains  was used to amplify the five selected genes. As a positive and a negative control of PCR of 5 genes, genomic DNA of M. intracellulare ATCC 13950T and distilled water were also used. The five primer sets were as follows: argH, argH_F 5’–TGA GCA AGT CCA CCC ATT TC– 3’ and argH_R 5’–TGG CGT CGA TGG AGT TGT C– 3’; dnaA, dnaA_F 5’–ACG AGC CTC AAC CGC C– 3’ and dnaA_R 5’–CTC ACG GCA CAG GTA CAT CG–R’; deaD, DEAD_F 5’–GGA ATA CAA GCA GGT GGC ACT– 3’ and DEAD_R 5’–GCG TTC GTA GTC CTG GAC CA– 3’; hsp65, hspF3 5’–ATC GCC AAG GAG ATC GAG CT– 3’ and hspR4 5’–AAG GTG CCG CGG ATC TTG TT– 3’ and recF, recF_F 5’–GAA ATC CCT GTC TGG CGC– 3’ and recF_R 5’–TCA TGC GCG CAT CTC C– 3’. Template DNA and each primer pair (20 pmol) were added to the PCR premix , and PCR was conducted by subjecting the samples to 5 min at 95°C, followed by 30 cycles of 95°C for 30 s, 58–60°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR reaction was performed in a MyCycler (Bio-RAD). The PCR products were detected and purified using the MEGAquick-Spin Total Fragment DNA Purification kit (iNtRON) for direct sequencing. Sequencing reactions were performed using an MJ Research PTC-225 Peltier Thermal Cycler and ABI PRISM BigDye Terminator Cycle Sequencing kits with the AmpliTaq DNA polymerase  following the manufacturer’s protocols. The obtained sequences were aligned using the MegAlign software package (DNASTAR), and then the SNPs in the sequences were analyzed.To confirm the different SNP patterns between the INT-5 strains and other M. intracellulare strains , 2 INT-5 strains (MOTT-H4Y and MOTT-36Y) and one M. yongonense strain (DSM 45126T) were analyzed using the genome sequence information  and the other including M. yongonense DSM 45126T and the two INT-5 strains, MOTT-H4Y and MOTT-36Y. This result indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126T than the 3 M. intracellulare strains  (The phylogenetic relationships between 3 ormation . All 6 sMOTT-64) .M. intracellulare ATCC 13950T, MOTT-H4Y, and M. yongonense DSM 45126T or M. intracellulare ATCC 13950T, MOTT-36Y, and M. yongonense DSM 45126T). At the level of 95% identity, M. intracellulare MOTT-36Y or MOTT-H4Y shared more orthologous coding sequences (CDSs) with M. yongonense DSM 45126T  than M. intracellulare ATCC 13950T   . This fiellulare .M. yongonense was previously reported to be based on the rpoB gene sequence. The sequence of this gene is identical to the distantly related species M. parascrofulaceum, which enables the separation of the 2 closely related species M. intracellulare and M. yongonense  -based phylogenetic analysis, the two INT-5 strains MOTT-H4Y and MOTT-36Y were clustered into the group including the M. intracellulare strains  and were separated from M. yongonense DSM 45126Tand M. parascrofulaceum ATCC BAA-614 , as shown in the genome sequence-based phylogenetic analysis , 2 INT-5 (MOTT-H4Y and MOTT-36Y), M. yongonense and M. parascrofulaceum showed that there were M. yongonense group- related SNPs in 17 genes  ] . DetailerpoB gene, there was no M. yongonense group-related SNPs, however, rpoB gene of M. yongonense shared identical 151 SNPs with that of M. parascrofulaceum. A concatenated phylogenetic tree was constructed using the extracted SNP sequences. The tree showed that the two INT-5 strains were clustered into M. yongonense DSM 45126T and separated from the other 3 M. intracellulare strains based on the phylogenetic analyses of the complete genome sequences and 35 concatenated gene sequences  were selected that possessed a higher number of M. yongonense group (M. yongonense DSM 45126T and two INT-5 strains MOTT-H4Y and MOTT-36Y) -related SNPs compared to the other genes. To explore the usefulness of this assay, sequence analysis of the five genes was applied to a total of 14 MAC clinical isolates from different Korean patients  and 7 was subjected to phylogenetic analysis.To develop SNP analysis to enable the selective identification of M. yongonense group-related SNPs in the partial argH gene sequence out of 10 M. yongonense group-related SNPs (from 105 nt to 657 nt). However, two INT-1 (Asan 29778 and 37721) and two INT-2 group (Asan 37016 and 38392) strains also shared one M. yongonense group-related SNP (G339C). All four INT-5 strains had 7 M. yongonense group-related SNPs in the partial dnaA gene sequence out of 7 M. yongonense group-related SNPs (from 627 nt to 1257 nt). However, one INT-2 group strain (Asan 38392) also shared one M. yongonense group-related SNP (C921T). All four INT-5 strains had 7 M. yongonense group-related SNPs in the partial deaD gene sequence out of 7 M. yongonense group-related SNPs (from 588 nt to 1191 nt). However, three INT-2 strains also shared one or four M. yongonense group-related SNPs . In the partial hsp65 gene sequence with 4 M. yongonense group-related SNPs (from 192 nt to 726 nt), two INT-5 group strains (Asan 36527 and Asan 36912) had only one M. yongonense group-related SNPs (G198A) and the three INT-1 or INT-2 SNPs , while the other two strains (Rhu and MOTT-68Y) had 4 M. yongonense group-related SNPs. All of the INT-5 group strains had 11 M. yongonense group-related SNPs in the partial recF gene sequence out of 11 M. yongonense group-related SNPs (from 520 nt and 1131 nt). However, one INT-1 (Asan 36309: T660C and G1123A) and one INT-2 strain (Asan 38392: T660C) shared one or two M. yongonense group-related SNPs.All four INT-5 strains had 10 M. yongonense may share were clearly separated from the other M. intracellulare clinical isolates  extracted from the five target genes showed that all four INT-5 strains of isolates . These rM. yongonense DSM 45126T than the M. intracellulare strains. This finding suggests the presence of another distinct genotype in M. yongonense that may not have been subjected to the LGT event of rpoB from M. parascrofulaceum. Therefore, M. yongonense could be divided into 2 distinct genotypes: one with the M. parascrofulaceum rpoB gene and the other with the M. intracellulare rpoB gene, depending on the presence of the LGT event of rpoB from M. parascrofulaceum  of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains, MOTT-H4Y and MOTT-36Y were more closely related to M. yongonense DSM 45126T, Asan 36912 and Asan 36527) belonging to the M. yongonense Type I genotype have been introduced via our 2 recent reports  may provide novel insights into our understandings regarding mycobacterial LGT mechanisms.LGT is the major mechanism by which bacteria can acquire genetic diversity, guaranteeing their survival under harsh environmental conditions , 32. HowargH, deaD, dnaA, hsp65 and recF) for the separation of M. yongonense from the closely related M. intracellulare strains. The concatenated 395-bp SNP-based phylogenetic analysis clearly separated 7 M. yongonense strains from 12 closely related M. intracellulare strains belonging to the INT-I and INT-2 genotypes, which were the first and the second most prevalent genotypes in Korean patients infected with M. intracellulare, respectively, with 83% bootstrap values  and 3 Type II strains   , suggestM. intracellulare should be revised to M. yongonense. Taken together, M. yongonense could be divided into 2 distinct genotypes depending on the presence of the LGT event of rpoB from M. parascrofulaceum: the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene. Additionally, we developed a novel SNP-based phylogenetic analysis to enable the taxonomic identification of M. yongonense clinical strains.In conclusion, our genome-based phylogenetic analysis indicated that the taxonomic status of the two INT-5 strains, MOTT-H4Y and MOTT-36Y previously identified as S1 Table(XLSX)Click here for additional data file.