Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing . This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of  Plasmodium. Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite  Plasmodium falciparum infections resistant to the former first-line drugs chloroquine (CQ) and sulfadoxine-pyrimethamine. Their impressive efficacy has been a major contributor to the 2-fold reduction in global malaria mortality achieved over the past decade, leading to calls for a global malaria elimination campaign predicated on the sustained efficacy of ACTs and mosquito vector control programs   monotherapy or an ACT, is now established in many areas of Southeast Asia –9. This –13 (K13) . A prima editing , 13. Rec editing –19. To dediting –, 20, 21,editing –. The preediting –, suggestbis-quinoline structure, comprised of two CQ-like 4-aminoquinoline moieties with a central linker, has led to the premise that PPQ, like CQ, acts via inhibition of heme detoxification, a critical process for P. falciparum blood-stage survival (––PPQ combined with the active ART metabolite dihydroartemisinin (DHA) is currently the first-line antimalarial drug in Cambodia and several neighboring countries, where it initially proved highly effective as a replacement for the failing drug combination of AS+MFQ . Mathemasurvival –28. Hemeurvival ––35.P. falciparum resistance to CQ is mediated primarily by PfCRT, a transmembrane protein present on the DV membrane whose mutant isoforms appear to efflux this drug out of the DV  of resistant parasites have been reported to be unreliable and do not necessarily capture differences between parasites in their dose-response profiles and rates of parasite survival following PPQ pressure  that would need to be both individually and collectively examined. Of note, a recent study identified those PfCRT variants only in parasites that were PPQ resistant in vitro  and blasticidin-S deaminase (bsd) selectable markers, respectively . Repeated attempts to achieve editing without short-term PPQ pulsing were consistently unsuccessful, presumably because of the poor growth that precluded expansion of edited lines in the absence of direct selection (see below).To generate the edited clones, we electroporated Dd2 parasites with the pDd2+C101F CL1 and Dd2Dd2+C101F CL2   . The recescribed  69)..Dd2+C101pfcrt, we performed whole-genome sequence analysis of the two Dd2Dd2+C101F clones and the recombinant control line Dd2Dd2. Illumina-based sequencing and analysis, conducted independently by two separate groups, confirmed the introduction of the C101F polymorphism into the pfcrt gene in both ZFN-edited Dd2Dd2+C101F clones. We also observed another mutation (R247S) in a gene (PF3D7_0912500) that codes putatively for an SAP domain-containing nucleic acid-binding protein. It would appear unlikely that this gene would contribute to PPQ resistance, and we suspect this is instead an unrelated mutation that arose spontaneously during the culture period. Importantly, no amplifications or deamplifications were observed in either Dd2Dd2+C101F clone, including at the pfmdr1 locus.To assess whether the 6 days of treatment with 40 nM PPQ selected for any genetic changes in addition to the introduction of the C101F mutation into C101F mutation. Dd2 and Dd2Dd2 parasites were electroporated on two separate occasions with the pZFN14/15 plasmid (used in our pfcrt gene editing transfections), but not the pcrtDd2-hdhfr donor plasmid used to edit pfcrt. We then applied 2 µg/ml of blasticidin (BSD) and 40 nM of PPQ pressure for 6 days. These selection conditions mirrored the ones used to generate the pfcrt-modified Dd2Dd2+C101F clones. None of the four transfections became positive within 80 days postelectroporation (compared to the Dd2Dd2+C101F transfections that became positive after 6 weeks), providing evidence that the 40 nM PPQ pulse did not independently select for resistant parasites but instead gave a slight advantage in selecting for Dd2Dd2+C101F parasites that were edited to carry the PfCRTC101F mutation introduced from the donor plasmid.We also carried out control transfection experiments as a separate approach to assess whether our use of short-term PPQ pressure might have selected for resistance independent of the PfCRTDd2+C101F, the cloned recombinant control line Dd2Dd2, and the unmodified parental Dd2 line were assessed for their in vitro susceptibilities to a panel of antimalarials. Initial susceptibility assays subjected parasites to a range of PPQ concentrations for 72 h, after which parasites were labeled with SYBR green I and MitoTracker Deep Red and parasitemias determined by flow cytometry. Results showed that both pfcrt-modified Dd2Dd2+C101F clones exhibited a highly PPQ-resistant phenotype with an ~140-fold shift in IC90 values compared to the Dd2Dd2 isogenic recombinant control   of the pfcrt-modified Dd2 lines. Download TABLE S1, PDF file, 0.1 MB.Mean ± SEM ICCopyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00303-17.7TABLE S2 Dd2+C101F line at different piperaquine concentrations. Download TABLE S2, PDF file, 0.04 MB.Representative parasitemias of the Dd2Copyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50) values using cytocidal assays, in which parasites were exposed to a range of PPQ concentrations for 6 h. The drug was then removed by extensive washing, and parasitemia was assessed 48 h later by flow cytometry. LD50 values were calculated by plotting the percentage of survival against log-transformed drug concentrations, using untreated parasites as the 100% survival benchmark. We observed a 3-fold increase in the LD50 level in both Dd2Dd2+C101F clones compared to the Dd2Dd2 control  PPQ LD50 and (B) LD90 values of the AMT-selected FCBC101F line carrying the PfCRTC101F mutation  Ratios of the IC50s  of the PfCRTC101F mutant lines (Dd2Dd2+C101F CL1 and Dd2Dd2+C101F CL2 and FCBC101F) compared to their Dd2 and FCB parental lines. IC50 data for each drug were generated from three independent 72-h dose-response assays performed in duplicate, as described previously  and the schizont stage (bottom panels) of development compared to the parental Dd2 line and the recombinant control, Dd2Dd2. Download FIG S3, TIF file, 1.4 MB.Morphology of the Dd2 Copyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro growth assays as a proxy measurement by comparing the in vitro replication rates of both Dd2Dd2+C101F clones to their isogenic control line, Dd2Dd2. These assays were carried out by measuring the rate of expansion of different parasite lines in one 48-h asexual blood-stage cycle after seeding them at 1% starting parasitemia . We observed a statistically significant, nearly 2-fold growth defect in the growth rate of parasites carrying the mutant pfcrtC101F allele (P < 0.01)  .in vitro drug susceptibility of the Dd2Dd2+C101F clones to a panel of registered antimalarial drugs, using standard 72-h assays. Results showed 9-fold and 14-fold increases in susceptibility to CQ and its active metabolite md-CQ, respectively, in the Dd2Dd2+C101F clones compared to parental Dd2Dd2 (P < 0.001)  . This inDd2+C101F clones compared to the recombinant control Dd2Dd2 (P < 0.001) , which are widely used ACT partner drugs. A nearly identical set of shifts in parasite susceptibility to these antimalarial agents was observed in both the Dd2Dd2+C101F and the FCBC101F lines (Results showed a 2-fold increased susceptibility to md-ADQ (the active metabolite of the 4-aminoquinoline ADQ), quinine , and ART (an endoperoxide) in the Dd2< 0.001) . No shif1F lines .50s was also observed in recombinant Dd2 clones harboring the C101F mutation compared to Dd2Dd2 (P < 0.01) (C101F parasites (50s of Dd2Dd2+C101F clones compared to Dd2Dd2 (P < 0.001). These findings highlight how mutant PfCRT isoforms are paradoxically selective yet pleotropic in modulating P. falciparum susceptibility to a wide diversity of antimalarial agents.A 35-fold increase in AMT IC < 0.01) , consistarasites . Our datC101F mutation in more mechanistic detail, we determined the effect of different concentrations of PPQ on Hz biocrystallization using a pyridine-labeled heme fractionation assay .In the presence of high concentrations of PPQ or CQ, we observed that the parasite’s ability to catabolize Hb was compromised, as shown by the statistically significant dose-dependent increase in undigested Hb observed in drug-treated Dd201F line  and D. I01F line . At simi10.1128/mBio.00303-17.4FIG S4 Dd2 and Dd2Dd2+C101F (clone 1). (A to C) Amounts of (A) hemoglobin Fe (femtograms per cell), (B) free heme Fe (femtograms per cell), and (C) hemozoin Fe present in Dd2Dd2 treated with increasing concentrations of PPQ. (D to F) Amounts of (D) hemoglobin Fe, (E) free heme Fe, and (F) hemozoin Fe present at increasing concentrations of PPQ-treated Dd2Dd2+C101F. (G to I) Amounts of (G) hemoglobin Fe, (H) free heme Fe, and (I) hemozoin Fe present in Dd2Dd2 treated with increasing concentrations of CQ. (J to L) Amounts of (J) hemoglobin Fe, (K) free heme Fe, and (L) hemozoin Fe present in Dd2Dd2+C101F parasites treated with increasing concentrations of CQ. Data are presented as means ± SEM . Statistical comparisons of the drug-treated lines to their untreated controls were performed using two-tailed Mann-Whitney U tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Download FIG S4, EPS file, 1.9 MB.Heme fractionation of PPQ- and CQ-treated Dd2Copyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00303-17.8TABLE S3 pfcrt-modified Dd2 parasites. Download TABLE S3, PDF file, 0.05MB.Hemoglobin, free heme, and hemozoin levels in drug-treated Copyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Dd2+C101F testing was extended to low LD50 multiples, because at 2× PPQ LD50 values we already observed exceptionally high levels of free heme, and cell recovery at higher PPQ concentrations was substantially reduced. With PPQ-treated Dd2Dd2 and Dd2Dd2+C101F, the results showed a statistically significant dose-dependent increase in free heme observed from 0.5× PPQ IC50 (22.2 nM) and upward in Dd2Dd2 and from 0.25× PPQ LD50 (40.6 nM) and upward in Dd2Dd2+C101F  was 32.1 ± 1.1 fg per cell, whereas that produced in Dd2Dd2+C101F was 46.7 ± 1.5 fg per cell at 2.5× LD50  and upward. The largest amount of free heme produced in CQ-treated Dd2Dd2 (observed at 3 × IC50) was 14.8 ± 1.0 fg free heme Fe per cell, which essentially matched the amount (15.2 ± 1.4 fg heme Fe per cell) observed at the equivalent CQ IC50 in Dd2Dd2+C101F  background, which differ in their expression levels of this gene. The FCB line expresses two copies, whereas the pfmdr1 knockdown FCB-KDmdr1 line expresses a single copy as a result of targeted disruption of the second copy  of the FCBmdr1-KD and FCB lines. Download TABLE S4, PDF file, 0.05 MB.Mean ± SEM ICCopyright © 2017 Dhingra et al.2017Dhingra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum asexual blood-stage parasites. This mutation, C101F, earlier observed in PPQ-pressured Dd2 parasites  exposure to PPQ. Of note, prior studies have provided conflicting evidence about the impact of traditional CQ-resistant mutant isoforms  on PPQ susceptibilities compared with CQ-sensitive parasites expressing wild-type PfCRT. Some reports with culture-adapted field isolates tested in vitro have found no difference   . By comppe pfcrt , 81. As pe pfcrt . Studiespe pfcrt . As a siin vitro . Interesast Asia . These mast Asia . Studiesaplotype . Gene-edpfmdr1 line and its 2-copy isogenic parent also provide evidence against pfmdr1 copy number having any impact on PPQ susceptibility  and how these could change drug binding to transporter substrate-binding sites  also saw a significant reduction in CQ IC50s . This plasmid also harbors the C101F mutation, which was introduced via site-directed mutagenesis  on pcrtDd2-hdhfr, using primers p1 and p2 (see pfcrt sequence is flanked by a pfcrt 5′ untranslated region (UTR) and a Plasmodium berghei crt 3′ UTR. Homology-directed repair (using pfcrt 5′- and 3′-UTR sequences as 5′ and 3′ regions of homology) resulted in integration of this modified pfcrt sequence, along with a downstream human dhfr marker that mediates resistance to the antimalarial agent WR99210  in RPMI 1640 with 11 mM glucose, supplemented with 2 mM l-glutamine, 25 mM HEPES, 2 g/liter sodium bicarbonate, 10 µg/ml gentamicin, 50 µM hypoxanthine, and 0.5% (wt/vol) AlbuMAXII (Thermo Fisher). Parasite cultures were maintained at 5% hematocrit at 37°C in an environment of 5% O2/5% CO2/90% N2. Transfections were performed by electroporating ring-stage Dd2 parasites at 5% parasitemia with 50 µg of purified circular plasmid DNA  to enrich for episomally transformed parasites. These parasites were then further transfected with pZFN14/15-bsd and selected with 2 µg/ml BSD (Thermo Fisher) for 6 days. We also applied 40 nM PPQ pressure for 6 days, beyond which the transfected cultures were selected only with 2.5 nM WR99210. Parasites were visible microscopically 4 to 6 weeks postelectroporation and were screened for editing , p5+p6 , and p6+p7 . Removal of introns from the edited Dd2 parasites results in slightly shorter PCR amplicons compared to the unedited Dd2. The presence of the PfCRTC101F mutation in these edited lines was confirmed by sequencing cDNA using primers p8 to p10  as described previously , >60.0; symmetric odds ratio (SOR), >4.0; Z score from Wilcoxon rank-sum test of alternative versus reference read mapping qualities (MQRankSum), <−12.5; and read positive rank sum (RPRS), <−8.0. Indels were filtered out if they met any of the following criteria: QD, <2.0; RPRS, <−20.0; FS, >200.0. Variants were annotated using snpeff (version 4.2) (The Nextera XT kit (Illumina) was used to prepare DNA libraries from samples for whole-genome sequencing using the dual-index protocol. The libraries were run on an Illumina HiSeq 2500 in the Rapid Run mode with 100-bp paired-end reads. The reads were aligned to the eviously , 102. Vaion 4.2) . Custom in vitro susceptibility of the Dd2Dd2+C101F clones to different antimalarials by comparing their IC50 and IC90 values to those of the recombinant isogenic control line Dd2Dd2 and the parental Dd2 line. IC50 and IC90 values were determined for PPQ, CQ, md-CQ, md-ADQ, QN, ART, LMF, MFQ, AMT, and BSD. md-CQ and md-ADQ are the in vivo metabolites of CQ and ADQ, respectively. To determine IC50s, we incubated parasites at 0.25% starting parasitemia and 1% hematocrit with a range of drug concentrations (across a range of 2-fold dilutions) at 37°C for 72 h in 96-well plates. Parasite growth in each well was assessed after 72 h using flow cytometry on an Accuri C6 cytometer with parasites stained with SYBR green I and MitoTracker Deep Red, as described previously  were made using two-tailed Mann-Whitney U tests on GraphPad Prism 6 software.We tested for changes in the eviously . In vitr50 values in Dd2Dd2+C101F and Dd2Dd2 clones were performed as previously described (g for 3 min), and the plates were incubated at 37°C for an additional 48 h. Parasitemias were then measured in each well using flow cytometry. The percentage of parasite survival (estimated as the ratio of parasitemia in drug-exposed to nonexposed wells) was curve fitted against log-transformed drug concentrations, using GraphPad Prism 6 software. Statistical comparisons of LD50 values were carried out using two-tailed Mann-Whitney U tests.Cytocidal assays to measure LDescribed  with sliC101F and FCB parasites  saponin and washed with 1× PBS (pH 7.5) to remove traces of the RBC Hb. RBCs and trophozoites were quantified in these samples using a hemocytometer and flow cytometry. The contents of the trophozoite pellet were then released by hypotonic lysis and sonication. Following centrifugation, the supernatants corresponding to membrane-soluble Hb  heme-pyridine complex was measured using a multiwell plate reader . The total amount of each heme species was quantified using a heme standard curve . The mas


Helicobacter pylori, colonize in stomach of ~50% of the world population. cag pathogenicity Island of H. pylori is one of the important virulent factors that attributed to gastric inflammation. Coinfection with H. pylori strain with different genetic makeup alters the degree of pathogenicity and susceptibility towards antibiotics. The present study investigates host immunomodulatory effects of H. pylori infection by both cag+ strain (SS1) and cag− strain (AM1). C57BL/6 mice were infected with AM1 or SS1 strain as well as AM1 followed by SS1 (AM1/SS1) and vice versa.+ T-regulatory (Treg) cells and immunosuppressive cytokines like IL-10 and TGF-β were reduced in AM1/SS1 compared to SS1/SS1 and SS1/AM1 coinfected mice gastric tissues.Mice infected with AM1/SS1 strain exhibited less gastric inflammation and reduced proMMP9 and proMMP3 activities in gastric tissues as compared to SS1/SS1 and SS1/AM1 infected groups. The expression of both MMP9 and MMP3 followed similar trend like activity in infected tissues. Both Th1 and Th17 responses were induced by SS1 strain more profoundly than AM1 strain infection which induced solely Th1 response in spleen and gastric tissues. Moreover, IFN-γ, TNF-α, IL-1β and IL-12 were significantly downregulated in mice spleen and gastric tissues infected by AM1/SS1 compared to SS1/SS1 but not with SS1/AM1 coinfection. Surprisingly, IL-17 level was dampened significantly in AM1/SS1 compared to SS1/AM1 coinfected groups. Furthermore, number of Foxp3H. pylori cag− strain infection attenuated the severity of gastric pathology induced by subsequent cag+ strain in C57BL/6 mice. Prior AM1 infection induced Th1 cytokine IFN-γ, which reduced the Th17 response induced by subsequent SS1 infection. The reduced gastritis in AM1/SS1-infected mice might also be due to enrichment of AM1- primed Treg cells in the gastric compartment which inhibit Th1 and Th17 responses to subsequent SS1 infection. In summary, prior infection by non-virulent H. pylori strain (AM1) causes reduction of subsequent virulent strain (SS1) infection by regulation of inflammatory cytokines and MMPs expression.These data suggested that prior  Helicobacter pylori, a class I carcinogen inhabit in the stomach of approximately 50% of the human population while only 10–15% population either develop chronic gastritis or gastric adenocarcinoma or gastric mucosa-associated lymphoid tissue lymphoma [H. pylori infection are poorly understood. However, accumulated evidences suggested that differences in host immune responses, environmental factors as well as the virulence properties of H. pylori strains may play important roles in determining the disease outcome. The most prominent H. pylori virulence-associated determinant is the cag pathogenicity island (PAI). It is a 40-kb genome segment that encodes the immunodominant protein cagA and type IV secretion system, which serve to transfer the bacterial cagA protein and other soluble factors such as peptidoglycans, to the cytoplasm of the host cells, known to play a key role in disease manifestation [cag PAI have been associated with more severe inflammatory responses than that induced by cag− strains [H. pylori specific host T cell response is predominantly a CD4+ T cell response polarized towards a T helper1 (Th1) phenotype [H. pylori induced inflammation are associated with the production of pro-inflammatory cytokines and appear to be triggered partly by genes located within the cag PAI [H. pylori infected subjects [H. pylori infection is also associated with a marked increased in cytokine IL-17 secretion from Th17 cells [H. pylori infection induced a mixed Th1/Th17 response [cagA and type IV secretion system are required for the induction of IL-17 responses in H. pylori infection [lymphoma . The undestation . Strains strains , 8, 9. Hhenotype . H. pylo cag PAI , 8, 12. subjects . Earlier17 cells . Involve17 cells . Recentlnfection . Secretinfection .H. pylori induced gastric inflammation with the upregulation of MMP9 and MMP3 in vivo [MMPs are a family of zinc dependent endopeptidases that play a crucial role in various pathological conditions including gastric ulcer –22. The  in vivo . MMPs ar in vivo .Heligmosomoides polygyrus, attenuated gastric pathology induced by H. felis [H. pylori infection attenuated Salmonella enterica serovar Typhimurium-induced colitis in C57BL/6 mice; this protective effect was associated with downregulation of the cecal Th17 response to S. typhimurium [H. pylori induced gastric pathology is also modulated by coinfection with other Helicobacter species. Coinfection of enterohepatic Helicobacter species (EHS), Helicobacter muridarum along with H. pylori attenuated the H. pylori induced gastric pathology in C57BL/6 mice [Helicobacter hepaticus with H. pylori lead to more severe gastritis as well as increased production of IL-17 cytokine [H. pylori also modulate gastric inflammation status [The gastrointestinal tract of human are colonized by various microorganisms which can be either commensalistic or pathogenic to human . The intH. felis . Attenuahimurium . Even seL/6 mice . Moreovecytokine . Interesn status .H. pylori infection but low level of gastric carcinoma incidence, widely known as African Enigma [H. pylori strains. We hypothesized that coinfection with a non pathogenic strain may provide protection against further infection of a virulent strain. The effect of coinfection on gastric inflammation, with different strains of H. pylori with or without cag Pathogenicity Island, has not been systematically studied yet. To investigate that, we established a coinfection in C57BL/6 mice using both cag− and cag+ strains of H. pylori and measured the gastric inflammatory pathways. We address whether prior cag− strain infection alleviate gastric damage induced by cag+H. pylori strain coinfection and the underlying host immunomodulatory mechanisms thereon. Here we for the first time documents that prior infection with cag−H. pylori strain dampens the disease severity for further cag+ coinfection.Surprisingly, in some African countries having lower economic status show high rate of n Enigma , 33. Thecag+ strains (SS1) and cag− strain (AM1) has the capacity to induce gastric inflammation, although the severity of damage was more pronounced in SS1 infection in C57BL/6 mice [H. pylori in different combination on disease burden of H. pylori induced gastric pathology in C57BL/6 mice. Different groups of mice were inoculated with vehicle for control, SS1 and boosted with SS1 strain, inoculated with AM1 boosted with AM1 strain, SS1 and boosted with AM1 strain and AM1 and boosted with SS1 strain of H. pylori separately and were sacrificed at day 10 post infection. Histological examination of mouse gastric tissues revealed that H. pylori infection in any combination of strains caused inflammation in gastric pit cells along with disruption in submucosa and muscularis mucosa compared with control  cells were detected in H. pylori induced gastric injury. Interplay among Th1, Th17 and Treg cells and their signature cytokines are crucial for H. pylori induced pathogenesis. To understand the role of inflammatory (Th17 and Th1) and immunosuppressive (Treg) cytokines in H. pylori induced gastric pathology, we compared the level of various cytokines in mice gastric tissues infected or coinfected with different strains of H. pylori. We found that H. pylori infection increased the expression of Th1 cytokines, TNF-α and IL-1β in mice gastric tissues  induced gastric pathology were significantly attenuated in mice that were earlier coinfected with cag− strain (AM1) and associated with modulation of Th17 and Treg cell responses.response , 6, 38. ysplasia , 39. Morpulation . Furtheresponses . In presnfection . AlthougH. pylori infection is associated with elevated Th1 cytokines [H. pylori induced gastric inflammation in mouse model and human [H. pylori infection and Th17/IL-17 pathway modulates Th1 cell responses [cag+ strain infection induced IL-17A secretion in mouse gastric tissues as well as spleen, while cag− infection did not. It seems to us that severe gastric inflammation in SS1/SS1 infected mice were mediated by both Th1 and Th17 responses while AM1/AM1 infection only by Th1 responses. We found that the level of Th1 cytokines IFN-γ, TNF-α and IL-1β in AM1/SS1 infected mice are comparable to SS1/AM1infected mice. In contrast, higher level of IL-17A was detected in SS1/AM1 mice than AM1/SS1 infected mice. Thus our results clearly indicate that attenuated gastric pathology in AM1/SS1 infected group is not due to reduced Th1 responses instead of reduced Th17 responses to AM1/SS1 infection. We conclude that the Th1 cytokine induced by prior AM1 infection particularly IFN-γ could also contribute in part to the downregulation of Th17 response induced by subsequent cag+ (SS1) infection because IFN-γ plays an inhibitory role towards Th17 cell activation [cag+ infection.It is reported that ytokines , 40. Hennd human , 41. Yunesponses . We fountivation , 43. ThuH. pylori infection have been postulated to be critically involved in the development of gastric ulcer. However, recent evidences suggest that apart from its well studied inflammatory and pathogenic functions, MMPs play a more complex and diverse role in ECM homeostasis, regulation of inflammation, arresting disease progression [H. pylori infection was significantly reduced when IL-17 is deficient or blocked [H. pylori, suggesting that cag PAI is required for the induction of IL-17 cytokine, and also indicates that the cells producing MMPs have responded to the increased IL-17 secretions. Our results also suggested that the reduced gastritis in AM1/SS1 infected mice may be is due to reduced activation of Th17/IL-17 pathway and subsequent downregulation of MMP9 and 3 expressions in AM1/SS1 infected group.Expression and secretion of different MMPs in gression . Role ofgression , 44. IL-+ Treg) cells suppress the host inflammatory responses during infection and thereby maintain physiological homeostasis of host immunity [H. pylori positive patients and H. pylori infected mice gastric tissues [H. pylori infected mice [+CD25+ Treg cells from H. pylori positive patients are more potent in the suppression of memory T cell responses [H. pylori induced gastritis was suppressed by adoptive transfer of Treg cells harvested from IL-10-competent C57BL/6 donor mice, demonstrating that IL-10-dependent Treg cells play a crucial role in suppressing H. pylori-induced gastric disease [+ cells as well as gastric IL-10 and TGF-β level were significantly higher in H. pylori infected mouse gastric tissues  supplemented with 7% sheep blood, 0.4% isovitalex and the antibiotics amphotericin B (8 µg/ml), trimethoprim (5 µg/ml) and vancomycin (8 µg/ml) (referred to here as BHI agar). The plates were incubated at 37 °C under 5% O2, 10% CO2 and 85% N2. In all experiments, overnight grown cultures on BHI agar plates were used.Two unrelated mouse adapted sed: SS1 , 35, andsed: SS1 . SS1 . Mice were divided into five groups (n = 6 in each), first group serves as control was given PBS only. Among the rest 4 groups 2 groups were orogastrically inoculated twice in a period of three days with either AM1 (cag+) or SS1 (cag−) strain (AM1/AM1 and SS1/SS1) and the rest 2 groups were given criss-cross infection. Criss–cross means infection by cag+ strain followed by cag− and vice versa (AM1/SS1 and SS1/AM1). Mice were sacrificed at day 10 after final inoculation (13 days post-primary inoculation).Male C57BL/6 mice with free access to food and water were obtained from institutional animal house. Experiments were designed to minimize animal suffering and to use the minimum number to obtained valid statistical evaluation. Animal experiments were carried out according to the guidelines of animal ethics committee of the institute. Animals of both control and experimental groups were fasted for 6 h with free access to water. . method . BrieflyGastric tissues of control and 10-day infected mice were sectioned for histological studies. The tissue samples were fixed in 10% formalin and embedded in paraffin. The sections (5 µm) were cut using microtome, stained with hematoxylin and eosin , and assg for 15 min and the supernatant was collected as PBS extract. The pellet was extracted in the lysis buffer  and centrifuged at 12,000×g for 15 min to obtain TX extracts. Tissue extracts were preserved at −80 °C for future studies.The pyloric part of the gastric mucosa of mice were suspended in PBS containing protease inhibitors, minced and incubated for 10 min at 4 °C. After incubation the suspension was centrifuged at 12,000×Blood samples were isolated from mouse by puncturing the heart followed by incubation for 30 min at room temperature. Serum was isolated from the clotted blood by low centrifugation. Serum sample was mixed with protease inhibitor mixture and stored at −80 °C. Equal volume of serum was used for gelatin zymography.For assay of MMP2, 9 and 3 activities, tissue extracts were electrophoresed in 8% SDS–polyacrylamide gel containing 1 mg/ml gelatin or casein (sigma) respectively, under non-reducing conditions. Seventy micrograms proteins were loaded in each lane. The gels were washed twice in 2.5% Triton X-100 (Sigma) and then incubated either in calcium assay buffer or in stromelysin assay buffer at 37 °C. Gels were stained with 0.1% Coomassie blue followed by destaining . QuantifFor assay of MMP9 activity in serum, mice serum samples were mixed with 1× nonreducing Laemmli sample loading buffer and were electrophoresed in SDS-8% polyacrylamide gel containing 1 mg/ml gelatin under nonreducing condition. Equal volume of serum samples were loaded in each lane. The gels were washed twice in 2.5% Triton X-100 and incubated in calcium assay buffer at 37 °C. Gels were then stained with 0.1% Coomassie Brilliant Blue stain followed by destaining. The zone of gelatinolytic activities appeared as negative staining. Quantification of zymographic bands were performed by densitometric analysis using Lab Image software .Helicobacter pylori infected and uninfected mice gastric tissues were homogenized in 1 ml sterile PBS, and centrifuged. The supernatants were analyzed for IFN-γ, IL-12, IL-17, IL-10 and TGF-β using sandwich ELISA kits  according to the manufacturer’s instruction. Total protein was measured by the Lowry method. The cytokines concentrations in gastric tissue extracts were expressed as picograms per milligrams of total protein [Helicobacter pylori infected and uninfected mice spleens were passed through meshed steel sieve to obtain single-cell suspension of splenocytes. Splenocytes (1.6 × 106 cells/ml) were cultured in RPMI 1640 medium with or without H. pylori whole cell protein (WCP) (2.5 μg/ml).The production of IFN-γ, IL-12 and IL-17 in the supernatants was measured by sandwich ELISA  after 48 h of splenocytes culture [For Immunofluorescence study, the tissue samples were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in ascending alcohol series . It was Tissue extracts (120 μg) were resolved by 10% reducing SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The memt test. P value less than 0.05 were considered as significant.Densitometry data are fitted using Sigma Plot. Data are presented as the mean ± SE. Statistical analysis was performed using the Student’s


Lactobacillus helveticus D75 and D76 were isolated from the intestinal tract of a healthy child. Both strains possess symbiotic, probiotic, and antagonistic activities. We have sequenced and annotated the whole genomes of L. helveticus D75 and D76 and have conducted a preliminary genome comparative analysis. Lactobacillus helveticus D75 and D76 have pronounced fermentative and probiotic activities (L. helveticus strains D75 and D76 to understand the mechanisms of regulation of their probiotic activity and syntrophic interactions.tivities . It was L. helveticus D75 and D76 were grown on de Man-Rogosa-Sharpe (MRS) medium  medium  at 37°C N50 values of 22,778 bp and 10,700 bp were obtained for L. helveticus D75 and D76, respectively. The HGAP algorithm in the SMRT Analysis pipeline version 2.3.0 was used to assemble the genomes of L. helveticus D75 and D76 from PacBio RS raw reads  –7. Genom–L. helveticus D75 and D76 were done using the Prokaryotic Genome Annotation Pipeline (PGAP) algorithm of the National Center for Biotechnological Information (NCBI) , with 1,693 protein-coding genes, 64 tRNA genes, and 15 rRNA genes. The total number of pseudogenes was 317. L. helveticus D76 includes 2,068 CDS, with 1,986 protein-coding genes, 64 tRNA genes, and 15 rRNA genes. The total number of pseudogenes was 265. The BLASTn algorithm was applied for preliminary genome comparative analysis of L. helveticus D75 and D76 with the genomes of L. helveticus DPC4571 and L. helveticus R0052, which were annotated and deposited in GenBank (L. helveticus D75 and D76 strains had 99.18% identity (90.65% coverage) and 97.73% identity (80.45% coverage) to the genomes of the DPC4571 and R0052 strains, respectively.The genome annotations of n (NCBI) . The ann GenBank , 11. TheL. helveticus D75 and D76 were 99.22% identical (with 76.3% coverage of the genome) to the genomes of L. helveticus species. Therefore, D75 and D76 strains, previously attributed to L. acidophilus species , were reclassified as L. helveticus D75 and D76.Calculating the average nucleotide identity (ANI) –14 showe–CP020029 (L. helveticus D75) and CP016827 (L. helveticus D76). The versions described in this paper are the first versions.These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession no.


As the use of Point of Care Testing (POCT) devices for measurement of glycated haemoglobin (HbA1c) increases, it is imperative to determine how their performance compares to laboratory methods. This study compared the performance of the automated Quo-Test POCT device (EKF Diagnostics), which uses boronate fluorescence quenching technology, with a laboratory based High Performance Liquid Chromatography (HPLC) method (Biorad D10) for measurement of HbA1c.Whole blood EDTA samples from subjects (n=100) with and without diabetes were assayed using a BioRad D10 and a Quo-Test analyser. Intra-assay variation was determined by measuring six HbA1c samples in triplicate and inter-assay variation was determined by assaying four samples on 4 days. Stability was determined by assaying three samples stored at −20 °C for 14 and 28 days post collection.2=0.969, P<0.0001). Mean (range) intra- and inter-assay variation was 1.2% (0.0–2.7%) and 1.6% (0.0–2.7%) for the D10 and 3.5% (0.0–6.7%) and 2.7% (0.7–5.1%) for the Quo-Test. Mean change in HbA1c after 28 days storage at −20 °C was −0.7% and +0.3% for D10 and Quo-Test respectively. Compared to the D10, Quo-Test showed 98% agreement for diagnosis of glucose intolerance (IGT and T2DM) and 100% for diagnosis of T2DM.Median (IQR) HbA1c was 60 (44.0–71.2) mmol/mol (7.6 (6.17–8.66) %) and 62 (45.0–69.0) mmol/mol (7.8 (6.27–8.46) %) for D10 and Quo-Test, respectively, with very good agreement (RGood agreement between the D10 and Quo-Test was seen across a wide HbA1c range. The Quo-Test POCT device provided similar performance to a laboratory based HPLC method. The analyser runs a single sample, requiring no user intervention following sample loading.The aim of this study was to compare the performance of the Quo-Test POCT analyser with a HPLC laboratory method.2HbA1c was determined using the BioRad D10 HPLC analyser  as the laboratory reference method and the Quo-Test POCT analyser . The BioRad D10 required 10 μL and the Quo-Test 4 μL of whole blood. Quality control was carried out every day of testing following the manufacturer's instructions using Biorad Lyphocheck Haemoglobin A1c Controls for the D10 and EKF Diagnostics Quo-Test A1c Controls for the Quo-Test.2.1Precision was determined by calculating the mean coefficient of variation (%CV) from six HbA1c samples assayed in triplicate for intra-assay variation and from repeat assaying of four samples on four days for inter-assay variation (range 34–72 mmol/mol for BioRad D10 and 33–104 mmol/mol for Quo-Test).2.2Whole blood EDTA samples (n=100) from subjects with and without diabetes were collected and assayed between June and August 2015. Ethical approval was obtained from the South East Wales Research Ethics Committee.2.3To test the stability of whole blood samples, aliquots from three samples were frozen immediately post measurement on day zero and stored at −20 °C. Thawed aliquots were measured on days 14 and 28 using both methods. The mean change in HbA1c over 28 days and the %CV  were calculated.33.1Mean (range) intra- and inter-assay CV for the D10 was 1.2% (0.0–2.7%) and 1.6% (0.0–2.7%), respectively and for the Quo-Test was 3.5% (0.0–6.7%) and 2.7% (0.7–5.1%), respectively.3.22=0.9691; p<0.0001).The comparison between D10 and Quo-Test is shown in 3.3Thawed aliquots that were measured on day 14 and 28 using both methods showed good stability. Over the period of 28 days the mean change in concentration from baseline for 3 samples was −0.7%  for the D10, and +0.3%  for the Quo-Test . Mean %C3.4According to WHO diagnostic ranges for HbA1c 4The purpose of this study was to compare the Quo-Test POCT analyser with an established laboratory method.Overall, there was very good agreement between the D10 reference analyser and the Quo-Test. The Quo-Test also gave results comparable to those given by the D10 after short term storage of whole blood samples at −20 °C. Previous studies have shown sample stability after short and long term storage at −70 °C for the measurement of HbA1c The Quo-Test, as a POCT analyser, has limitations compared to an HPLC method such as the inability to measure HbA1c outside the working range of 20–140 mmol/mol 4–15%). Also, as with any other HbA1c analyser, the result obtained using the Quo-Test is dependent on the patient's clinical history and may be affected in patients who have an abnormal typical lifespan of their blood cells, for example haemolytic anaemia Despite the improvements in performance of POCT compared to laboratory analysers, the use of POCT HbA1c measurement in diagnosis of diabetes is not currently supported in clinical guidelines 5The performance of the Quo-Test POCT analyser is similar to that of a laboratory HPLC analyser. The Quo-Test POCT analyser should be considered for diagnostic purposes by the various professional organisation that issue clinical guidelines.


The receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells.+CD8+ cytotoxic T cells, CD49b+NK cells, CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+ MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8+ T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-β1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively.Melanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3+ and CD8+ T cells. The percentage of CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-β1.Downregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4These findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-β1. Malignant melanoma, one of the most highly aggressive tumors, resists to conventional chemotherapy and radiotherapy and has fatal outcomes. There are compelling evidences to show that melanoma cells escape the host’s immunity by actively developing multiple suppressive mechanisms within the cancer microenvironment . For insThe Notch signaling is a highly conserved pathway that controls the differentiation, development and function of multiple cell types, such as stem cells . MammalsAlthough Notch signaling is known to be important for the malignant biological behavior of melanoma cells, little is known about the effects of aberrant activation of this pathway in melanoma on tumor-induced immunosuppressive microenvironment. Our primary study has shown that siRNA-mediated Notch1 knockdown might potentially enhance the effect of IL-2 immunotherapy in malignant melanoma . In the 2.Murine malignant melanoma cell line B16 was purchased from China Center for Type Culture Collection. B16 cells were cultured in DMEM-high glucose  supplemented with 10% fetal bovine serum  at 37 °C in an atmosphere of 5% CO5 B16, B16-shNotch1 or B16-Notch1 melanoma cells were subcutaneously inoculated. Mice were observed carefully while tumor volume in mice was measured twice a week. Tumor volume was calculated by (length × width2)/2. All mice were sacrificed humanely after the experiments. Melanoma tissues in mice were surgically resected to perform further assay. Animal care and treatment were in accordance with institutional guidelines. All animal study protocols were reviewed and approved by the Animal Care and Use Committee of Southern Medical University, China.Female C57BL/6 and BALB/c Nude mice were purchased from Laboratory Animal Center of Southern Medical University . All mice were 6- to 8 weeks of age at the time of experiment, and at least 6 mice per group were used in each experiment. For tumor challenge experiments, 5 × 10Lentivirus containing mouse Notch1 (lentivirus-mNotch1) and Notch1 shRNA (lentivirus-shNotch1) were constructed by gene technology company . The day before infection, cells were plated at a density of 20~30% in a good condition. The lentivirus above (containing fluorescence) were used to transfect B16 cells according to the manufacture’s protocol. The culture medium was changed to normal medium 12 h after infection. Three days later, the cells were subjected to fluorescence activated cell sorting (FACS) with flow cytometer for further experiments.5) were co-cultured with 30 Gy-irradiated B16 (5 × 104) cells in 96-well plates precoated with mouse IFN-γ for 20 h at 37 °C in complete DMEM medium in triplicate. The cell suspension was aspirated and washed with deionized water. Then Biotinylated anti-mouse IFN-γ antibody was added and incubated for 2 h at room temperature. After washing with deionized water, a streptavidin-horseradish peroxidase solution was added and incubated for 1 h at room temperature. Then an aminoethyl carbozole substrate solution was added and incubated for 15 min. Spots in plate were counted using a stereomicroscope after washing.Mouse IFN-γ ELISpot kits  were used according to the manufacturer’s instructions. Briefly, immune cells from spleens or tumor-DLNs  and then fixed and permeabilized with a permeabilization buffer . Cells were finally stained with antibody to IFN-γ or FoxP3 for 50 min at 4 °C, washed again, and analyzed by FACSCalibur . Irrelevant IgG mAbs were used as a negative control. Ten thousands live events were acquired for analysis.At the indicated time points, tumor draining lymph nodes(tumor-DLNs) and spleens were harvested from the mice, and minced into small fragments and mechanically dispersed in 3-5 ml cold PBS. After filtering with 70 μm cell strainer , Single-cell suspensions were adjusted to 1 × 10+T, CD4+T and NK cells before and during treatment with lentivirus-shNotch1, the transplanted mice received intravenous injections of 0.3 mg mAb from anti-CD8+ hybridoma  the anti-CD4+ hybridoma  or 0.5 mg of antiasialo-GM1 antibody . Antibody injection was started on the day of tumor innoculation, and the treatment was repeated every 5 days throughout the entire experimental period to ensure the depletion of the target immune cell subset.To deplete CD8+ T lymphocytes were purified using microbead isolation kits followed by magnetic-activated cell sorting (MACS) according to the manufacturer’s instructions . Isolated cells were resuspended at a concentration of 1 × 106 cells/mL in RPMI-1640 medium  containing L-glutamine (2 mM), penicillin/streptomycin (100 U/mL), and 10% fetal calf serum .Splenic lymphocytes were isolated from splenic cell suspension using density gradient centrifugation . CD8+T cells and B16 cells. B16 cells were plated in lower chamber with 104 cells per well. CD8+T lymphocytes were then added to the upper chamber in 1:1 ratio. For CFSE analysis, CD8+T cells were labelled with CFSE staining before co-cultured with B16 cells. After co-cultured for 48 h, CD8+ cells in upper chamber were harvested for CCK8 assay or analysis of CFSE dilution and apoptosis.3.0 μm Transwell chamber were used for co-culture of CD8+ T cells isolated from B16 tumor-bearing mice were cultured for 3 days with irradiated B16 stimulators in a complete 1640 RPMI medium. The responder cells were then collected and used as effector cells in a 8 h chromium release assay against indicated B16 target cells. B16 target cells were labeled by combining 5 × 106 cells with 50 μCi 51Cr (Perkin-Elmer Japan Co.) in a total volume of 0.2 ml complete RPMI for 1 h at 37 °C, followed by washing thrice with plain RPMI. For the chromium release assay, CD8+ effector cells were mixed with B16 target cells at the different ratio of 1:1, 5:1 and 25:1 in a 96-well round-bottom plate (BD Biosciences).CD85 cells/ml. The culture supernatants were harvested after B16 cells had adhered to the bottom of the culture plate for 18h. Then, the supernatants were centrifuged to remove melanoma cells. In accordance with the manufacturer’s instructions, Culture supernatants of B16 cells were measured by ELISA for the secretion of TGF-β1, VEGF and IL-10 . Freshly excised tumor tissues were minced and homogenized for 1 min in tissue tearor. Then, supernatants were collected after centrifugation and subjected to ELISA for examining the secretion of TGF-β1, VEGF, IL-10 and IFN-γ using mouse ELISA kits according to the manufacturer’s protocol .B16 cells were seeded into 6-well plates at a density of 2 × 10P < 0.05. All statistical analyses were performed using SPSS 20.0 .All data were expressed as means ±SEM. Results of tumor volume, flow cytometry, Western blotting, ELISA, inhibition rate on lymphocyte proliferation and in vitro cytotoxicity were assessed using a student’s t-test. Differences were considered to be statistically significant when To examine the role of Notch1 in tumor growth, we stably silenced Notch1 expression using shRNA in B16 tumor cells Fig. . B16-shNIn order to confirm this finding, we intravenously inoculated B16-shCon, B16- shNotch1, or B16-Notch1 melanoma cells into C57BL/6 mice. The number of lung tumor foci in C57BL/6 mice was counted on day 14 after intravenous inoculation. Pulmonary metastasis in mice receiving B16-shNotch1 cells was significantly inhibited, whereas pulmonary metastasis was enhanced in those receiving B16-Notch1 melanoma cells Fig. .To explore whether regulation of tumor growth by Notch1 expression in melanoma cells was due to host immune status, we subcutaneously inoculated B16- shCon, B16-shNotch1, or B16-Notch1 melanoma cells into BALB/c Nude mice, which are deficient in T, B and NK cells. In contrast to immunocompetent mice, no significant differences of tumor burden were observed in BALB/c Nude mice challenged with B16-shNotch1 or B16-Notch1 cells Fig. . The res+ or CD8+ T cells or NK cells and tumor growth was monitored. CD8+ T cell depletion significantly compromised the effect of Notch1 expression on tumor growth. However, the compromised effect of CD4+ T cells or NK cells depletion was less important than CD8+ T cells in Notch1-mediated tumor growth (Fig. + T cells in immune system.To further determine how Notch1 affected the antitumor immunity, B16-shNotch1 or B16-shCon cells were subcutaneously inoculated into C57BL/6 mice that were depleted of CD4wth Fig. . These r+CD8+ CTL and NK cells in tumor-DLNs (Fig. + T cells and IFN-γ-expressing NK cells (Fig. To investigate the effect of Notch1 in tumor tissue on local anti-tumor immunity, we analyzed the immune status in the tumor microenvironment of B16 tumor-bearing mice. We found that knocking down Notch1 remarkably reduced while overexpressing Notch1 increased the percentage of CD3LNs Fig. . As IFN-lls Fig. . In contlls Fig. . These r+CD8+ CTL and NK cells in splenocytes (Fig. We next analyzed the spleen T lymphocytes to evaluate the system immunity. Knocking down or overexpressing Notch1 did not affect the percentage of CD3tes Fig. . Then, Ites Fig. . Consisttes Fig. . In cont+ and CD4+ T cells in tumor-DLNs were analyzed by flow cytometry. Knocking down Notch1 reduced PD-1 expression while overexpressing Notch1 showed an increased PD-1 expression on CD8+ and CD4+ T cells (Fig. − melanoma cells in tumor (Fig. The PD-1/PD-L1 axis is a critical regulator of T cell fate and function. PD-1 has also been identified as a marker of T cell exhaustion, a hypo-functional cell state found in tumor. The expression of PD-1 on CD8lls Fig. . No signmor Fig. . Tregs amor Fig. .Fig. 5No+ T cells isolated from B16 tumor-bearing mice were expanded and activated with irradiated B16 cells in culture for 3 days prior to test cytotoxic function against B16-shNotch1 or B16-Notch1 cells by chromium release assays. The cytotoxicity of T cells against B16-shNotch1 was more powerful than that against B16-shCon cells (Fig. To explore the mechanism of immunosuppression induced by Notch1 in B16 cells, splenic CD8lls Fig. . The dimlls Fig. .Fig. 6No+T cells isolated from wild-type B16 tumor-bearing mice were co-cultured with Notch1 knocked down or knocked in B16 cells in transwell chamber for 48 h. The CD8+T cells were cultured in the upper chamber and B16 cells were in the lower chamber. According to microscopic data, knocking down Notch1 showed more lymphocyte colony formation than control group (Fig. +T cells which were co-cultured with B16-shNotch1 cells (Fig. To evaluate the role of Notch1 in B16 cells on inhibiting T lymphocyte proliferation, CD8oup Fig. . In CCK8lls Fig. . The efflls Fig. . Moreovells Fig. . In contlls Fig. . These r+T lymphocyte apoptosis (Fig. To study how Notch1 affect local anti-tumor immunity in vivo and vitro, mRNA expression and supernatant secretions of TGF-β1, VEGF and IL-10 in B16 cells were measured by Q-PCR and ELISA, respectively. Notch1 knocking down remarkably reduced, while overexpression of Notch1 showed significant increase in mRNA expression and supernatant secretion of TGF-β1 Fig. . Howeversis Fig. , prolifesis Fig.  and actisis Fig.  induced To determine the secretion of immunosuppressive cytokines and IFN-γ in vivo, homogenate of tumor tissue from B16 tumor-bearing mice were harvested for ELISA assay. The data showed that TGF-β1 secretion in tumor tissue was significantly reduced in B16-shNotch1 tumor, while increased in B16-Notch1 tumor in vivo Fig. . HoweverMalignant melanoma is a potentially fatal cancer characterized by rapid progression, metastasis to regional lymph nodes and distant organs, and resistance to chemotherapy and radiotherapy . MelanomAmong immune suppressor cells, Treg cells and MDSCs are significantly increased in hosts with advanced malignancies. It is well established that Treg cells and MDSCs are essential for the control of autoimmune responses, and that their accumulations in tissues or peripheral blood of tumor-bearing mice are responsible for the suppression of anti-tumor immune effector T cell functions . Cancer + cytotoxic T lymphocytes infiltration and IFN-γ release in tumor tissues. It could also up-regulate PD-1 expression on CD4+ and CD8+ T cells and increased the ratio of CD4+CD25+FoxP3+ Treg cells and CD11b+ MDSCs in tumor microenvironment. All these contributed to weakened anticancer immune responses and promoted tumor growth.In the present study, Notch1 expression in melanoma cells upregulated TGF-β1 mRNA expression and promoted TGF-β1 secretion in tumor microenvironment, which enhanced tumor-mediated inhibition of T cell proliferation and activation. Furthermore, Notch1 inhibited CD8Interaction between Notch and TGF-β signaling has been identified in multiple tumors to regulate a wide variety of complicated tumor biological behaviors. For example, heightened Dll4/Notch signaling in tumor cells can magnify TGF-β-induced pSMAD2/3 signaling and is required to sustain TGF-β-induced tumor cell growth . Moreove+ cytotoxic T lymphocytes infiltration and higher IFN-γ release in tumor microenvironment, while tumor growth was significantly suppressed. This revealed a more powerful function of CD8+ cytotoxic T cells and more intense anticancer immune responses in vivo. The opposite effects were found in Notch1 overexpression cells. All these results strongly suggested that aberrantly elevated TGF-β1 secretion in response to abnormal activation of Notch signaling was a potential mechanism of melanoma-induced immunosuppression.TGF-β is produced by tumor cells and tumor-infiltrating immune cells within the tumor microenvironment. The role of TGF-β in immune suppression includes the inhibition of T cell proliferation, cytokine production and cytotoxicity , 25, as In conclusion, we found that Notch1 signaling in melanoma cells facilitated tumor immune escape and promoted cancer progression via TGF-β1 secretion. Inhibition of Notch1 expression in melanoma cells might be considered as a potential therapeutic method for melanoma immunotherapy.


PIP3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PIP2. The PTEN tumor suppressor is thought to function primarily as a PIP3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PIP2 3-phosphatase, both in vitro and in vivo. PTEN is a major PIP2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PIP2 4-phosphatase, leads to synergistic accumulation of PIP2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PIP2 levels in a mouse model of prostate cancer, and it inversely correlated with PIP2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PIP2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PIP  •2 3-phosphatasePTEN is a PIP•2 accumulation downstream of class I PI3KPTEN and INPP4B regulate PIP•2-dependent activation of Akt and formation of invadopodiaPTEN regulates PIP•2 signaling may play a role in the tumor suppressor function of PTENPIP 2 3-phosphatase within the growth factor-stimulated PI3K signaling network, in addition to its accepted role as a PIP3 3-phosphatase. This suggests that specific PIP2 effector functions, such as invadopodia formation, play a role in the PTEN-loss-of-function phenotype.Malek et al. show that the tumor suppressor PTEN acts as a PIP PIP3 is retained in the lipid bilayer and promotes the translocation and/or activation of a variety of effectors that recognize the head group of this lipid with appropriate affinity and specificity. The best studied of these effectors are the PH domain-containing serine/threonine protein kinases PDK-1 and AKT1/2, which indirectly regulate the mTORC1 complex and promote anabolic growth and survival P2 and PIP2, respectively. The best-studied 3-phosphatase is PTEN, and loss-of-function mutants cause significant elevations in PIP3 in various cell and animal models P3 levels in leukocytes and other tissues, but recent studies suggest other 5-phosphatases may also play a role, depending on cell context Pl models . The maj context .3 is also unclear, as is the physiological significance of dephosphorylation via either route. Removal of the 5-phosphate from PIP3 produces PIP2, and recent evidence points to additional signaling roles for this lipid. PIP2 can bind to some effectors with similar affinity to PIP3, for example, AKT or DAPP1, but specific roles for this lipid have also been defined, particularly in the regulation of actin mesh-works associated with endocytic structures, lamellipodia, and podosomes/invadopodia Padopodia . Furthern of AKT .3 and PIP2 signals generated by activation of class I PI3Ks is current technical limitations in their quantitative measurement. Historically, the most accurate method for measuring these lipids has been radiolabelling, lipid extraction, deacylation, and separation of the relevant head groups by anion-exchange high-performance liquid chromatography (HPLC), which is laborious, requires substantial amounts of radiolabelled precursors, and cannot be applied to tissue biopsies. Recently, we described an approach to measure PIP3 by HPLC-mass spectrometry (MS) that circumvents many of these issues, but this method does not distinguish between regio-isomers of the same mass, for example, PIP2 and PIP2 PIP2 .2 and PIP2. We then applied this and existing methods to execute a systematic screen for the impact of gene deletion and/or small interfering RNA (siRNA) suppression of known phosphoinositide phosphatases on shaping PIP3 and PIP2 signals in response to growth factor stimulation. These and follow-up studies led us to discover that PTEN acts as a major PIP2 3-phosphatase, in addition to its known role as a PIP3 3-phosphatase. Moreover, deletion of PTEN in a mouse model of prostate cancer results in very high levels of PIP2 accumulation in hyperplasic epithelial cells, revealing another mechanism by which PTEN acts as a tumor suppressor.We describe here an extension to our MS method, which allows HPLC separation and measurement of PIP2 and PIP2. We have now developed a modification to this method based on ozone-catalyzed cleavage of C = C double bonds to reduce acyl chain length P2 found in mammalian tissues  to allow an independent estimate of their relative amounts P2 in our biological extracts (We have previously shown that methylation of the acidic phosphate groups of polyphosphoinositides with trimethylsilyl (TMS)-diazomethane allows sensitive detection of these molecules by HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MS) . This men length A. Chroma amounts B and 1C.latelets B, a resplatelets . Furtherextracts A and S1B3 and PIP2. Initially, we employed an siRNA screen directed against all phosphoinositide phosphatases previously reported to act on these two lipids and expressed at significant levels in these cells  cells (PTEN−/− isogenic cell line (PTEN-knockout [KO]) P2, and PIP3 in selected starved and EGF-stimulated cells.We disrupted expression of phosphoinositide phosphatases in Mcf10a cells, and then we used our new and existing HPLC-MS methods to measure the effect on epidermal growth factor (EGF)-stimulated accumulation of PIPT) cells A. We alsut [KO]) A. We the3 (2 (2 (3 response to EGF were knockdown (KD) or deletion of PTEN or SHIP2 P3 levels (3- to 4-fold), though in each case the PIP3 response remained transient, with levels falling within 5–15 min of EGF stimulation P3 A, a smal3 B, and ba3 (2 B and PI(3 (2 (2 C. These 3 (2 (2 . The onlor SHIP2 A and S2Amulation A. These 2 (2 and PIP pools (data not shown), and, therefore, they probably result from an indirect effect on acyl composition.We also note that loss of PTEN caused an apparent drop in the levels of PIP2 C and a s2 B. Howeve2 under either starved or EGF-stimulated conditions P3 in the same cells and amounting to ∼20% of the total PIP2 pool Pnditions B. This wan cells . Howevere to EGF B. The peIP2 pool A–2C.2 in these cells compared to our analogous siRNA knockdown studies P studies B. Furtheudies  auto-phosphorylation was very similar across the relevant knockdown and knockout cell lines C. Furthe3 and PIP2 pools in WT, PTEN-KO, SHIP2-KD, SHIP2-KD,PTEN-KO, and INPP4A/B-KD,PTEN-KO cells. EGF stimulated clear accumulation of the PIP3 reporter in the proximity of the plasma membrane in all cells examined P2 antibody Pexamined A, and EGKO cells B. Furtheantibody C.Figure 2 have been suggested, including 5-dephosphorylation of PIP3, 3-phosphorylation of PI4P by class I PI3Ks, 3-phosphorylation of PI4P by class II PI3Ks, or sequential phosphorylation from PI3P by unidentified kinases P2 in our system.Several alternative pathways for cell surface receptor-stimulated accumulation of PIP kinases . We used50s). These molecules inhibited the EGF-stimulated accumulation of PIP3 and PIP2 in INPP4A/B-KD,PTEN-KO cells with very similar potency P2 accumulation to PI-103 and the PI3Kα-selective inhibitor BYL-719 P3. Surprisingly, given that we had demonstrated an involvement of SHIP2 in regulating PIP3 levels, siRNA knockdown of INPP4A/B caused similar accumulations of PIP2 in PTEN-KO cells and PTEN-KO cells in which SHIP2 expression had also been deleted by CRISPR/Cas9, though possibly with slightly slower kinetics P3 (caused by the loss of both SHIP2 and PTEN). We therefore investigated the effect of multiple knockdowns of known 5-phosphatases in INPP4A/B-KD,PTEN-SHIP2-KO cells. Of the combinations examined, only combined knockdown of SHIP1, SYNJ1/2, INPP5F, and INPP5B caused significant elevations in PIP3 and a modest reduction in PIP2 Pkinetics B. We reaIP2 E.2 produced on EGF stimulation may be derived directly by class I PI3K-mediated 3-phosphorylation of PI4P, rather than indirectly via 5-dephosphorylation of PIP3. To estimate the flux through PIP3 dephosphorylation, we added 1 μM PI-103 1 min after adding EGF, to prevent further activity of class I PI3K, and then we monitored the fall in PIP3 levels P3 maintained a high rate of PIP3 dephosphorylation P3 levels B. PIP2 dephosphorylations P2, PIP2, PI3P, and PI4P Pylations  and S2. and PI4P C. We parand PI4P . Parametand PI4P  and S2. 3 is 1.4:1, suggesting a substantial fraction of PIP3 is recycled back to PIP2 upon class I PI3K activation P3 being available to 5-phosphatases  support PIP2 production with a relative flux of ∼2.65:1 Ptivation D. When Pphatases D. EGF-st ∼2.65:1 D. The ob2 dephosphorylation, together with a further unknown phosphatase (Y). Simulations show that PTEN must act directly on PIP2 and not merely increase the levels of PIP3 and thence flux through its 5-dephosphorylation. In INPP4A/B-KD cells, all PIP2 dephosphorylation flux is routed through PTEN and the unknown phosphatase Y, with a relative flux of approximately 16:1 P2 in INPP4A/B-KD,PTEN-SHIP2-KO cells in the presence of PI-103 Pely 16:1 E. The mof PI-103 , howeverf PI-103 .2 in starved, unstimulated cells without invoking a separate pool of PIP2 that is both insensitive to EGF and is not synthesized by class I PI3K P2_BG; 33P]-Pi-radiolabelling Pabelling . The res2 phosphatase and provides around 57% of the total PIP2-phosphatase activity. PTEN has previously been shown to be a poor PIP2 phosphatase in assays with recombinant protein and simplified lipid substrates P3 or d6-PIP2 substrates (to avoid any ambiguity as to the origin of products derived from these substrates).A clear prediction of our mathematical model is that PTEN acts as a direct PIPbstrates . We re-e3 at both 3- and 5- positions, producing d6-PIP2 and d6-PIP2, respectively P3 3-phosphatase active under these conditions. Cytosol from cells lacking SHIP2 produced similar amounts of d6-PIP2 to cytosol derived from cells containing SHIP2, consistent with the conclusion above that Mcf10a cells contain multiple PIP3 5-phosphatase activities.Under assay conditions resulting in less than 10% consumption of substrate, cytosol from WT cells dephosphorylated d6-PIPectively A and 5B.2, generating d6-PIP, at a very similar rate to cytosol from WT cells P2 3-phosphatase PWT cells C. RemarkWT cells C. The deant PTEN B, demons see the , and thisphatase A.2, but this might be predicted from our lack of understanding of the relative efficiency with which PIP3 and PIP2 activate AKT.Previous work has shown deletion of PTEN or INPP4B results in hyperactivation of AKT . We comp2 also has a separate, specific role in mediating Tks5-dependent formation of actin-rich structures called invadopodia Padopodia . These sadopodia . We measadopodia C and 6D.2 phosphatase in vitro. To extend these observations to an in vivo context in which PTEN has been shown to play an important role, we investigated the prostate epithelial cell-specific deletion of PTEN in a mouse model of prostate cancer Pe cancer . In the 2 and PIP3 accumulation by HPLC-MS in prostate biopsies taken from Ptenflox/flox,PbCre−/− (WT) or Ptenflox/flox,PbCre+/− (PTEN-KO) mice at 10 weeks of age  mice at 16 weeks of age P2 in whole-prostate biopsies Ps of age A and 7B.s of age C and secs of age D and 7E.16 weeks E. PIP2 in PTEN-KO versus INPP4B-PTEN-KO prostates P2 levels suggested there may be lower expression of INPP4B in mouse prostate compared to Mcf10a cells. We therefore compared the relative expression levels of PTEN and INPP4B in several mouse tissues and Mcf10a cells by western blotting. PTEN expression was similar across all samples analyzed, but expression of INPP4B was very variable, with high expression in mouse brain Prostates D and S7Bse brain  and Mcf1se brain F. The lo2 and PIP3 in a range of human prostate and breast cancer cells and also relative expression of INPP4B and PTEN P2. Two exceptions were T-47D and HCC-1187, which had normal expression of PTEN but large PIP3 responses. T-47D has previously been shown to possess an H1047 gain-of-function mutation and increased copy number of PI3KCA . Clearly, there will be many factors at play in determining the flux through 5-dephosphorylation of PIP3, including levels of active receptor, and thus a comparison between a relatively small number of cancer cells, with multiple differences in mutational status and gene expression, is difficult. Nevertheless, these results broadly support the idea that PTEN is likely to be a widespread regulator of PIP2 levels but that this role may be more apparent in cells with reduced expression of INPP4B.We measured PIPand PTEN C–S7F. Lof PI3KCA . HCC-118f PI3KCA . The cel2 accumulation is something that clearly requires further investigation.Interestingly, prostate lines lacking PTEN exhibited relatively reduced responses to IGF-1 E. We sus3 and PIP2 signals produced in response to EGF. While our results confirm the importance of PTEN and SHIP2 as phosphatases that regulate the accumulation of PIP3, they also point to a complex picture in these cells that is most easily explained by significant compensation among multiple phosphatases for the loss of any one individual enzyme. The extent to which these compensatory mechanisms reflect shared roles under normal conditions, are simply driven by the mass action effect of a rise in PIP3, or depend on additional activation mechanisms is unclear. Significant redundancy among 5-phosphatases may also explain the lack of prevalent tumor suppressors among this family, though the recent identification of PIPP (INPP5J) as a potential tumor suppressor in breast cancer points to contexts in which individual enzymes may predominate P2 production lags behind that of PIP3 P2 accumulation suggest it is not necessary to invoke this explanation.We systematically screened for phosphoinositide phosphatases in Mcf10a cells that can selectively shape the PIPdominate . We also3,4,5)P3  and our 2 3-phosphatase in Mcf10a cytosol. This is surprising given that careful in vitro studies have previously suggested that PIP2 is a very poor substrate for PTEN, at least compared to PIP3 P2 dephosphorylation, to form PI3P P2, and deletion of INPP4B alone had no discernible effect. However, combined deletion of PTEN and INPP4B had a very large, synergistic effect on EGF-stimulated PIP2 accumulation, suggesting that in the cellular environment these two enzymes can each compensate effectively for the other’s absence with respect to PIP2 hydrolysis.We clearly demonstrate that PTEN is an active PIP3,4,5)P3 . Furtherorm PI3P . Phosphoorm PI3P , and we 2 produced by class I PI3K at the plasma membrane can be dephosphorylated by PTEN to form PI4P has been underestimated. This conclusion is supported by studies showing overexpressed PTEN can influence PIP2-reporter distributions in lymphocytes  or in different locations  is likely to be controlled by different phosphatases, and, at least in some of these contexts, the dephosphorylation of PIP2 by INPP4A/B to form PI3P is likely to predominate Pphocytes , and it phocytes . It is aposition . It is idominate .2 levels; in 10-week-old prostates the levels of PIP2 rose from undetectable to approximately 50% of PIP2. Deletion of INPP4B alone had an insignificant effect on PIP2 accumulation, and combined deletion of INPP4B and PTEN had an insignificant impact above deletion of PTEN alone. Previous work has shown that deletion of PTEN in mouse prostate drives class I PI3K-dependent epithelial cell hyperplasia and tumor growth with 100% penetrance Pnetrance , and, th2, a conclusion supported by our analysis of a limited collection of breast and prostate cancer cell lines. Direct evidence has been presented for a context-dependent role for INPP4B as a tumor suppressor, with deletion of Inpp4b driving tumor formation in mouse thyroid only in the absence of one allele of Pten P of Pten . A simpl of Pten  suggests cancers . Further cancers . Our res2 accumulation in PTEN-INPP4B-KO cells suggests the flux through 5-dephosphorylation of PIP3 is surprisingly large and that the steady-state levels of PIP3 are dynamically regulated by very fast rates of synthesis and degradation. Whether 5-dephosphorylation brings a quantitative or qualitative element to the class I PI3K-signaling network has been debated, with strong evidence now presented for both roles P2 effectors TAPP1/2 P2-dependent negative feedback, reducing activation of AKT and sensitizing cell growth to PI3Kβ inhibitors, an effect suggested to result from direct PIP2-mediated inhibition of class I PI3Ks Pth roles . The losroles P2 in cellular extracts and the properties of recombinant PTEN in the in vitro assays constructed to date. In the context of class I PI3K activation, loss of PTEN raises the levels of PIP3, increases flux through PIP3 5-phosphatases, and slows dephosphorylation of the resulting PIP2. In some tissues, loss of PTEN alone is sufficient to drive huge accumulations of PIP2 . In other cells, combined loss of both PTEN and INPP4B is required to see equivalent increases in PIP2 . These very large accumulations of PIP2 will distort class I PI3K pathway signaling, through both a quantitative effect on the activation of common PIP2 and PIP3 effectors  and a signaling imbalance through the activation of PIP2-selective effectors . The effects of this distortion will be context dependent, but, for PTEN-dependent tumorigenesis and metastasis, the contribution of PIP2-specific processes is clearly an area that now demands further investigation. The potential role of PTEN as a PIP2 phosphatase under normal physiological conditions, within both class I and class II PI3K-signaling pathways, is also a concept that now warrants further attention.Our results reveal an important and widespread role for PTEN as a PIPphillip.hawkins@babraham.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Phillip Hawkins  and ‘PTEN-KO’  mice and backcrossed to the C57BL/6J strain for at least 4 generations; these mice were housed in the Biological Support Unit at The Babraham Institute. INPP4B−/− mice have been described previously  mice; these mice were housed in the Akita University Animal House.PB-Cre4 mice  and PTENoxP mice  have beeeviously  and wereNo estimation of simple size was performed as sample sizes were not chosen based on pre-specified effect size. Instead, multiple independent experiments were carried out using several biological replicates specified in the legends to figures.Prostates from several age-matched mice of identical genotype were analyzed.Male mice were used.The animals were kept under SPF conditions and the animal facilities where the mice were kept were regularly checked for standard pathogens. Health reports can be provided upon request.Prostates were prepared from mice not subject to any previous experimentation.Mice used for all experiments were naive. No drug tests were done.All animal experiments at The Babraham Insitute were reviewed and approved by The Animal Welfare and Ethics Review Body and performed under Home Office Project license PPL 70/8100. Animal experiments in Akita were reviewed and approved by the Akita University Institutional Committee for Animal Studies, Akita University. The mice were looked after by professional caretakers. Every animal was checked daily.2 containing bedding and nesting material. Food and water was provided ad libitum. The light cycle ran from 6 am to 6 pm.Animals housed in the Biological Support Unit at the Babraham Institute were kept under specific pathogen–free conditions. Mice in the animal facility in Akita were kept in groups of up to six animals in standard IVC cages of 524 cm−/− Mcf10a cell lines were generated by targeted homologous recombination and were obtained from Horizon Discovery Ltd together with their parental cell lines. All Mcf10a cell lines were maintained at 37°C with 5% CO2 in DMEM/F12 supplemented with 5% horse serum, 10 ng/mL EGF, 10 μg/mL insulin, 0.1 μg/mL cholera toxin, 0.5 μg/mL hydrocortisone, 1% w/v penicillin/streptomycin (complete medium). Starvation medium consisted of DMEM/F12 supplemented with 1% charcoal/dextran treated fetal bovine serum, 0.1 μg/mL cholera toxin, 0.5 μg/mL hydrocortisone, 1% P/S.Mcf10a cells are non-transformed human female breast epithelial cells. PTENHuman prostate cancer cells  and breast cancer cells  were obtained from the AstraZeneca cell bank and had been previously authenticated using DNA fingerprinting short tandem repeat assays. All revived cells were used within 10 passages and cultured at 37°C with 5% CO2 for less than 2 months. Benign prostatic hyperplasia epithelial cell line BPH-1 was cultured in RPMI-1640 supplemented with 20% FBS, 10 μg/mL insulin, 6.7 ng/mL sodium selenite, 5.5 μg/mL transferrin, 0.5 nM dihydrotestosterone and 1% w/v penicillin/streptomycin. The remaining prostate and breast cancer cell lines were grown in RPMI-1640 with 10% FBS and 1% w/v penicillin/streptomycin.8/mL in HEPES–Tyrode buffer  supplemented with 0.1% [w/v] D-glucose, 10 μM indomethacin and 0.02 U/mL apyrase, and allowed to rest at 30°C for 30 min prior to experimentation.Venous blood was obtained from a healthy female human volunteer with the approval of the local research ethics committee at the University of Bristol, UK. The donor provided written informed consent, and reported as not having taken medication in the 14 days prior to donation. Blood was drawn into 4% trisodium citrate , and acidified with acidic citrate dextrose . Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 180xg for 17 min at room temperature. Platelets were subsequently pelleted by centrifugation of the PRP at 650 x g for 10 min at room temperature in the presence of 10 μM indomethacin and 0.02 U/mL apyrase (grade VII). Platelets were resuspended at 4 × 108 platelets were preincubated with DMSO or 100 nM Wortmannin for 10 min, before treatment with HEPES–Tyrode buffer or 0.5 U/mL thrombin for 3 min under stirring at 1200 r.p.m using a Chronolog 490-4D aggregometer at 37°C . Treatment was stopped by the addition of 750 μl ice-cold 1 M HCl and samples were centrifuged at 12000 x g for 10 min at 4°C. Resulting pellets were frozen until lipid extraction.1 × 105 cells were seeded per 35 mm dish, and were subject to reverse transfection (using transfection agent DharamaFECT1) with a pool of 4 different siRNA  in Optimem and 10% complete medium, according to manufacturer’s instructions. Media was changed after 16 hr and replaced with complete medium for 32 hr, after which cells were washed with dPBS and maintained in starvation media for 16 hr. Cells were stimulated with 10 ng/mL of EGF for the indicated times. Where indicated, cells were pre-incubated with inhibitors for 20 min prior to EGF stimulation. Stimulations were terminated by aspiration of media and washing with ice-cold PBS, prior to processing of the cells for lipid or western blot analysis as described below.1.6 × 10https://chopchop.rc.fas.harvard.edu/ or http://crispr.mit.edu/ and cloned into all-in-one pSpCas9(BB)-2A-GFP plasmid, plasmid as described previously . Retrovirus was generated by transfecting 10 μg pMIGR1-mCherry-PH-TAPP into amphotropic phoenix cells  using lipofectamine 2000, according to the manufacturer instructions. Following 24 hr incubation, media was replaced with complete Mcf10a medium and incubated at 32°C for a further 24 hr. Retroviral containing media was then collected and passed through a 0.45 μm filter, before adding to WT or PTEN4P2O7, 5 mM β- glycerophosphate, 1 mM Na3VO4, 25 mM NaF). After 30 min solubilisation at 4°C with agitation, lysates were centrifuged  and the supernatants collected and diluted in SDS-PAGE sample buffer. Proteins  were resolved on a SDS-PAGE gel, transferred to PVDF membranes and blotted with the indicated primary antibodies at 4°C overnight. They were then washed in TBS  containing 0.1% v/v Tween 20 and incubated with a mix of Infrared Dye coupled secondary antibodies. The membranes were imaged with the Li-Cor Odyssey Infrared Imaging System using the 700 nm and 800 nm channels. Signals were quantified using the Image Studio software. Alternatively, membranes were washed and incubated with HRP-conjugated secondary antibodies and signals detected using the ECL detection system. Signals from the HRP-incubated membranes were quantified using Aida software.Cells were scraped and lysed in 150 μL of ice-cold lysis buffer . 1x reducing SDS sample buffer  was pre-warmed to 70°C and 750 μL of sample buffer was added per 50 mg tissue to yield an approximate final protein concentration of 4 mg/mL. Lysates were homogenized by vortexing for 15 s followed by a sequential syringe step through a 21G needle (3x), followed by a 23G needle (3x). Proteins were denatured by boiling at 95°C for 5 min. Lysates were cleared by centrifugation for 5 min at 20,238 x g, after which the syringe and centrifugation steps were repeated. Proteins were resolved by SDS-PAGE  and immunoblotted for the indicated antibodies.Tissues were pulverized under a continuous flow of N2, PIP3 measurement, 250 μL for PIP2/PIP2 measurement, and the remaining cells were kept for analysis by western blot. Cells were pelleted in a microfuge , the supernatant removed and cell pellets either processed immediately or snap-frozen in liquid nitrogen and stored at −80°C for up to two weeks.750,000 Mcf10a cells grown on a 35 mm dish were killed in 750 μL ice-cold 1 M HCL, then scraped and collected into an Eppendorf tube. Each sample was then split into three separate 2 mL polypropylene Eppendorf tubes; 250 μL for PI, PIP, PIPFor human prostate and breast cancer cell lines, 250,000 cells were seeded into 35 mm dishes and grown in the medium optimal for each cell line for 32h. Cells were then starved for 16h by replacing the medium with starvation medium – a phenol red-free RPMI 1640 supplemented with 2mM glutamax. Following stimulation and / or inhibition with appropriate reagents, medium was removed by aspiration and cells killed with 750 μl ice-cold 1M HCL. Cells were then scraped, collected into Eppendorf tubes, pelleted, and snap-frozen, as described above.3:H2O(acidic) was added to the cell pellets and the mixture vortexed thoroughly for 10 s. Relevant internal standards were then added:10 ng C17:0/C16:0-PIP3, 100 ng C17:0/C16:0-PI, 250 ng d6-C18:0/C20:4-PIP2 for routine analysis of PI, PIP, PIP2 and PIP3; 50 ng C17:0/C20:4 PI, 50 ng d6- C18:0/C20:4-PIP2, 250 ng d6-C18:0/C20:4-PIP2 for routine analysis of PI, PIP2 and PIP2. Lipids were then extracted using an acidified Folch phase partition and derivatised with TMS-diazomethane (920 μL of a solvent mixture containing 2:1:0.79 (v/v) MeOH:CHClomethane .2, and PIP3 were measured by HPLC-MS P2 (see below). The C38:4 species of PIP2 and PIP3 represent approx. 10%–15% of the total species of these lipids in Mcf10a cells and all species behave very similarly upon stimulation with EGF P2 were analyzed by a new HPLC-MS method, see below. Response ratios were calculated for the endogenous C38:4 species of these lipids divided by their relevant d6-labeled internal standard. In some experiments, absolute amounts of these lipids were generated by reference to standard curves Pd curves . Three t2 gas, thus care should be taken). We added post-derivatisation wash solution (700 μl) to the organic solution, and mixed the samples, which we then centrifuged , and collected the resultant lower phase. We repeated the wash step, and then added MeOH:H2O  to the final collected lower phase. The samples were then dried down under a stream of nitrogen at room temperature without warming until dry. 160 μL methanol was then added and sonicated briefly, then left at RT for about 30 min prior to ozonolysis. A C-Lasky ozone generator was used in the following procedure. The unit was set to use air as the oxygen source at a flow rate of 4 dm3/min and the flow was split after the ozone generator so that approximately 75 to 90% of the flow went to an ozone destruction unit and the remaining fraction was used to bubble through the solution containing the samples. The power level on the ozone generator was set to about 60% of the maximum level. The Ozonolysis procedure started by placing the glass sample vials containing the methylated lipid solutions in an aluminum block which was cooled in an acetone/dry ice bath to a temperature of about −70°C. Ozone was then bubbled through the solutions for 5 min. Dimethyl sulphide (2 μl) was then added to each sample and then allowed to warm up to RT. Water (40 μl) was then added to each sample which was then ready to be submitted for analysis by UPLC, using the following conditions for PI2/PI2 separation:Solvent A: Water, 0.1% formic acidSolvent B: (40% acetonitrile/60% methanol), 0.1% formic acidColumn temperature: 60°CInjection volume 45 μlLipids were extracted and derivatized with TMS-diazomethane; we added 2 M TMS-diazomethane in hexane (50 μl) to lipid extracts prepared as described above (approx I mL of ‘lower phase’), to give a yellow solution, and then allowed the reaction to proceed for 10 min, RT. We quenched reactions with glacial acetic acid (6 μl), which removed the sample’s yellow color P2 and PIP2 described above, except for the following UPLC-MS condtions:Column: ACE Excel 2 C18-Amide, 150 mm x 0.5 mmSolvent A: Water, 0.1% formic acidSolvent B: (40% acetonitrile/60% methanol), 0.1% formic acidColumn temperature: 60°CAdditional sample preparation: Take 40 μl of sample prepared as above and add to 200μl 70% methanol /30% water. Inject 5 μlGradient:Mass spectrometer parameters as above.Transitions:We added 0.1 mL of 1.5 M NaCl to 1 mL of [33]P-Pi and then diluted this mixture into a phosphate-depleted medium (GIBCO) and supplemented with  to reach a final concentration of 250 μC/mL. After siRNA transfection, as described above, cells were starved for 16 hr, then washed twice with phosphate-depleted medium before adding the [33]P-Pi–containing medium for 90 min. Then we stimulated the cells with 10 ng/mL hEGF. We aspirated the medium then stopped the reaction with ice-cold 1 M HCl. We extracted and deacylated the lipids, and analyzed the glycerophoinositides by strong anion-exchange chromatography .6) were seeded in 10 cm tissue culture dishes and grown for 64 hr, then washed twice with ice-cold PBS prior to lysis with 1 mL of lysis buffer ) on ice. Cells were then scraped on ice, collected in 2 mL safe lock Eppendorf tubes, vortexed, and kept on ice for 5 min. The lysate was sonicated on ice  and then ultracentrifuged . A 50 μL aliquot was taken for subsequent protein analysis and this, along with the remainder of the sample, were snap frozen in liquid N2 and stored at −80°C. Protein determination was performed on the 50 μL aliquots. The remainder of each sample was then thawed and all samples normalized to the same protein concentration with lysis buffer. The lysates were then divided into 100 μL aliquots and frozen at −80°C. For in-lysate phosphatase assays, a 100 μL aliquot was thawed on ice, then an aliquot containing 4 μg of protein was diluted to 20 μL with a solution containing 2 mg/mL of BSA, 160 mM KCl, 1 mM MgCl2, 20 mM HEPES pH7.3 and 1 mM EGTA. These 20 μL aliquots of cytosol were then used directly in the phosphatase assays, see below.Mcf10a cells P2 and either d6-C18:0/C20:4-PIP2 or, d6- C18:0/C20:4-PIP3, was prepared in a glass vial at 32.4:23.2:10.5:23.7:2.6:0.9:2.9:3.7 (w/w) ratio  and solvents evaporated under vacuum. In order to reconstitute the lyophilized lipid mixture into micelles, 200 μL of a solution containing 20 mM HEPES pH7.3, 0.1 mM EDTA and 1 mM EGTA was added and the lipid solution bath sonicated for 1 min and divided into 20 μL aliquots.A 20 μL aliquot of diluted cell lysate was then added to 20 μL micellar solutions and incubated at 30°C for the indicated times. Reactions were terminated by the addition of 1 mL ice-cold 20% trichloroacetic acid (TCA), followed by centrifugation  and a single wash with 5% TCA. Samples were then incubated on ice for 5 min, centrifuged  and the supernatant aspirated. The resultant pellet was then processed for lipid analysis as described for cell pellets, see above.4 cells were seeded in complete medium on the fluorescent gelatin surface and left to adhere (between 2-2.5 h). Next, cells were washed with PBS, starved for 4 hr, and then stimulated with 20 ng/mL hEGF for another 6 hr. Both complete and starvation media were supplemented with 10 ng/mL rhTGF-β1. After washing with PBS and fixing in 3.7% PFA (15 min at RT), cells were labeled for cortactin following a previously described protocol . For IHC, prostates were rinsed in PBS and fixed in 4% paraformaldehyde for 1 hr at room temperature. Prostates were then cryo-protected by immersion in 30% w/v sucrose in PBS, while rotating at 4°C, for 1 hr for WT prostates, and for 2-3 hr for PTEN-KO prostates. Prostates were then immersed in embedding medium and slowly frozen on dry ice. Embedded prostates were stored at −80°C until use. 12 μm cryosections were prepared on charged glass slides using a Leica CM1850 cryostat. INPP4B-KO and PTEN-INPP4B-KO prostates were dissected intact and prepared for measurements of phosphoinositides or for immunofluorescence on site at Akita University.Mice were sacrificed using Schedule 1 methods, in agreement with the Animals (Scientific Procedures) Act 1986 (ASPA) and tissues rapidly dissected. Prostates, consisting of anterior, ventral, and dorsolateral lobes (one pair of each lobe), were dissected intact and one set of anterior, dorsolateral, and ventral lobes was used for western blot, while the other set was used for IHC. For western blot and measurements of phosphoinositides, tissues were rinsed in PBS and flash-frozen in NH&E staining of prostate cryosections prepared on glass slides was performed using Mayer’s hematoxylin and Eosin Y solutions, following a standard protocol. Images were acquired using a Zeiss Laser Microdissection microscope (20x or 40x air objectives), stitched with AxioVision4 software (5% overlap) and gaps automatically filled with Adobe Photoshop. Alternatively, an Olympus BX41 microscope equipped with a 40x oil objective was used to obtain higher resolution images. These were then manually stitched using Velocity software with a brightness correction.12 μm mouse prostate cryosections prepared on glass slides were stained for PIP2 by permeabilising the sections with saponin . Cells were labeled with the anti-PIP2 antibody at 1:150 dilution for 2 hr at room temperature, then sections were washed 3 times with PBS and incubated with streptavidin-Alexa Fluor 674 for 1 hr at room temperature, in a humid dark room. In some experiments, an additional incubation with anti-phospho-Akt-S473 antibody was carried out according to the manufacturer’s instructions . After having washed the sections three times with PBS, sections were incubated in PBS supplemented with Hoechst dye for 10 min; after 3 washes with PBS, sections were then mounted with a hard-set medium. Sections were imaged with a wide field Nikon Live Cell Imager microscope and 20x air objective. Multiple fields of view were stitched automatically with 10% overlap using NIS-Elements software integrated with the microscope.Unpublished work from our laboratories had indicated that the most predominant molecular species of PIP3 and PIP2 which accumulate in mouse prostate on deletion of PTEN were the shorter chain, more saturated species. We therefore used a new HPCL-MS method (manuscript in preparation) to analyze 17 different species of phosphoinositides in this tissue and present results for the combined total of all species measured . Data were corrected for wet weight of tissue. At least three individual mice were used per genotype.A range of mathematical models were built and analyzed in COPASI . The mod∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. For the invadopodia assay in Mcf10a clones (Unless stated otherwise, data are means ± SEM of at least three biological replicates, a clones D, Tukey’A strategy for randomization, stratification or blind selection of samples has not been carried out. Sample sizes were not chosen based on pre-specified effect size. Instead, multiple independent experiments were carried out using several sample replicates as detailed in the figure legends.Conceptualization, P.T.H. and L.R.S.; Methodology, A. Kielkowska, J.C., and T.S.; Software, V.Y.K., P.P., and N.L.N.; Investigation, M.M., A. Kielkowska, T.C., K.E.A., D.B., P.P., H.N., S.E., A. Koizumi, J.S., V.J., A.G., A.V., D.S., M.I., and N.L.N.; Resources, I.N., S.C., and T.S.; Writing – Original Draft, P.T.H., L.R.S., A. Kielkowska, M.M., and T.C.; Supervision, P.T.H., L.R.S., D.S., S.C., N.L.N., T.H., T.S., and S.F.; Funding Acquisition, P.T.H., L.R.S., S.B., T.S., T.H., and S.C.