extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
PTEN opposes negative selection and enables oncogenic transformation of pre-B cells

PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role of PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small molecule inhibition of PTEN in human pre-B ALL cells resulted in AKT hyperactivation, p53 checkpoint activation and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-BCR signaling, which engaged a deletional checkpoint for removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for elimination of autoreactive B cells and represents a new strategy to overcome drug-resistance in human ALL.

Introduction
The majority of newly generated pre-B cells in the bone marrow are eliminated at the pre-B cell receptor (pre-BCR) checkpoint 1 . Critical survival and proliferation signals come from the pre-BCR: If pre-B cell clones fail to express a functional pre-BCR, signaling output is too weak. If the pre-BCR binds to ubiquitous self-antigen (autoreactive immunoglobulin μ heavy chain; μ-HC), pre-BCR signals are strong. Both attenuation below minimum (e.g. non-functional pre-BCR) and hyperactivation above maximum (e.g. autoreactive pre-BCR) thresholds of signaling strength trigger negative selection and cell death. Approximately 75% of newly generated pre-B cells express an autoreactive μ-HC 2-3 , highlighting the importance of stringent negative selection of autoreactive clones at the pre-BCR checkpoint. While autoreactive pre-B cell clones are eliminated owing to the toxicity of strong pre-BCR signaling [1][2][3] , sustained activation of Phosphoinositide 3-kinase-AKT (PI3K-AKT) signaling is sufficient to rescue B cell survival in the absence of a functional BCR 4 and required for pre-B cell survival 5 . Likewise, germline mutations in humans that result in either loss or hyperactivation of PI3K-AKT signaling have equally deleterious effects on human early B cell development 6 , suggesting that early B cells are selected for an intermediate level of PI3K signaling.
Phosphatase and tensin homolog (PTEN) is a key negative regulator of the PI3K-AKT pathway and functions as a dual protein and lipid phosphatase, which dephosphorylates PtdIns(3,4,5)P 3 (PIP3). PTEN counteracts PI3K, which phosphorylates PtdIns(4,5)P 2 to generate PIP3, the membrane anchor and ligand of the AKT-PH domain 7 . Deletions or inactivating mutations of PTEN are frequently observed in all main types of human cancer (on average 8.3% among 37,898 samples studied) 8 . The common outcome of these lesions is increased membrane levels of PIP3 and AKT-hyperactivation. Genetic lesions of PTEN mutations also play a major role in hematopoietic malignancies. For instance, lesions in PTEN, PI3K and AKT pathway component genes are present in up to 50% of T cell lineage ALL cases 9 .

Pten is required for initiation and maintenance of pre-B ALL in vivo
To study a potential role of PTEN and negative regulation of PI3K-AKT signaling, we developed BCR-ABL1and NRAS G12D -driven models of pre-B ALL (Fig. 1). To this end, we transformed IL7-dependent pre-B cells from bone marrow of Pten fl/fl and Pten +/fl mice 10 with oncogenic BCR-ABL1 or NRAS G12D . BCR-ABL1 represents the driver oncogene in Ph + ALL, the most common subtype of pre-B ALL in adults (~30%). RAS pathway lesions affect NRAS, KRAS, PTPN11 and NF1 and occur in ~50% of both adult and pediatric ALL 11 . Together, BCR-ABL1 and NRAS G12D driven pre-B ALL reflect two major non-overlapping types of human ALL (~80%). For inducible deletion of Pten, pre-B ALL cells were transduced with tamoxifen (Tam)-inducible Cre-ER T2 (Cre) or ER T2 (estrogen receptor) empty vector (EV) controls. After selection of the transduced cells with puromycin, Cre was activated by Tam (1 μmol/l), which induced excision of LoxP-flanked Pten alleles and depletion of PTEN protein within two days (Fig. 1a). Notably, inducible Cre-mediated deletion of Pten in pre-B ALL cells resulted in rapid cell death of leukemia cells (Fig. 1b, Supplementary Fig. 1a). To address whether loss of PTEN not only affected survival of established leukemia but also leukemia-initiation, we reversed the order and first induced deletion of Pten in IL7-dependent Pten fl/fl pre-B cells and subsequently induced BCR-ABL1-mediated transformation. Two days after induction of Cre, Pten +/+ and Pten −/− pre-B cells were transduced with GFP-tagged BCR-ABL1. Pten +/+ pre-B cells showed rapid outgrowth of BCR-ABL1 GFP -expressing clones, indicating leukemic transformation. In contrast, Pten −/− pre-B cells carrying BCR-ABL1 GFP were not susceptible to malignant transformation by BCR-ABL1 ( Fig. 1c; Supplementary Fig. 1b). These findings were recapitulated in an in vivo transplant setting. BCR-ABL1-transformed Pten fl/fl pre-B ALL cells caused fatal leukemia in transplant recipient mice within 18 days. Luciferase bioimaging revealed that Cre-mediated deletion of Pten did not interfere with engraftment of pre-B ALL cells. However, pre-B ALL cells failed to initiate fatal disease in the absence of PTEN and transplant recipients survived for indefinite periods of time (Fig. 1d). Minimal residual disease (MRD) analysis by genomic PCR revealed no trace of covert leukemia clones ( Supplementary Fig. 1c).

Pten mediates feedback regulation of pre-BCR and its co-receptor CD19
To elucidate the mechanism of how the tumor suppressor PTEN, seemingly paradoxically, enables oncogenic transformation of pre-B cells, we studied gene expression changes upon inducible Pten-deletion (Fig. 1e). Loss of Pten induced expression of multiple markers of lymphocyte activation including Cd80, Cd86, Cd25 (Il2rα) and Ly6a (Sca-1). Pten-deletion resulted in downregulation of the IL7 receptor, both at the mRNA level and surface expression (Fig. 1e, f). In addition, multiple components of the pre-BCR (Ighm, Vpreb1, Syk) and its co-receptor CD19 were all downregulated upon deletion of Pten ( Fig. 1e-f). In pre-B cells, PI3K-AKT signaling is initiated from the pre-BCR via Syk 4-5,12-13 and CD19 via recruitment of PI3K 14,15 to a YXXM motif in the cytoplasmic tail of CD19. For this reason, loss of pre-BCR and CD19 expression in response to deletion of Pten suggests PTEN-mediated feedback regulation of these PI3K-AKT activating receptors. Likewise, B cell precursors developing in Mb1-Cre Pten fl/fl mice lost CD19 expression upon early deletion of Pten in the B cell lineage (Fig. 1g). Western blot analysis confirmed near complete loss of CD19 protein expression upon deletion of Pten, demonstrating loss of both surface and intracellular expression of CD19 (Fig. 1h).

Deletion of Pten compromises BCR-ABL1 and NRAS-driven leukemogenesis
BCR-ABL1 and NRAS G12D -driven ALL subtypes were dependent on PTEN function to a similar degree as inducible, Cre-mediated deletion of Pten induced cell death and reduced colony forming ability ( Fig. 2a-b). Genotyping of colonies revealed that Pten fl/fl pre-B ALL clones that were able to form colonies had retained Pten flox alleles despite activation of Cre.
Hence, these few colonies arose from pre-B ALL clones that had evaded Cre-mediated deletion of Pten ( Supplementary Fig. 2).
Pten-deletion also induced G0/G1 cell cycle arrest and senescence in pre-B ALL cells ( Fig.  2c-d). To assess the effect of acute ablation of Pten in fully established BCR-ABL1 and NRAS G12D -driven pre-B ALL cells in vivo, 100,000 Pten fl/fl pre-B ALL cells carrying tamoxifen-inducible Cre were injected into sublethally irradiated NOD/SCID recipient female mice. 5 days after transplantation of Pten fl/fl pre-B ALL cells, leukemia-initiation and engraftment was confirmed by bioluminescence imaging and Cre was induced by 10 consecutive daily injections of tamoxifen. Consistent with in vitro results, Pten-deletion caused leukemia regression in vivo (Fig. 2e) and prolonged overall survival in the recipient mice. We conclude that Pten is required for both initiation and maintenance of pre-B ALL in vivo.

Pre-B ALL cells are exempt from genetic lesions of PTEN
Given the unexpected sensitivity of pre-B ALL cells to even a moderate dose-reduction of Pten, we reassessed the concept of PTEN as a tumor suppressor in human cancer and leukemias and lymphomas. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency of mutations in solid cancer (8.3% in 37,898 samples) and hematological malignancies (8.4% in 2,548 samples) 8 . In addition to point mutations, deletions of PTEN at chromosome 10q23 are frequent in cancer (5.3% in 8,071 samples studied; Fig. 3a). These findings are in agreement with previous mechanistic studies demonstrating a tumor suppressor role of PTEN in acute myeloid 16 and chronic myeloid leukemia (CML) 17 , in T cell lineage acute lymphoblastic leukemia 9,[18][19] , and in mature B cell lymphoma [20][21][22] . However, point mutations in PTEN were not detected in a single case of 694 pre-B ALL patient samples. Likewise, no PTEN deletions were found in 231 pre-B ALL cases (Fig. 3a). Similarly, while oncogenic activation of the PI3K-AKT pathway in leukemia and lymphoma also occurs through activating mutations of agonists of the PI3K-AKT pathway, pre-B ALL cases do not harbor such mutations ( Supplementary Fig. 3). Together, these genetic data suggest that PTEN lesions and other mutations that lead to oncogenic activation of the PI3K-AKT pathway are not favorable in pre-B ALL and that PTEN may have a fundamentally different role in pre-B ALL compared to other hematopoietic malignancies.

High expression levels of PTEN in patient-derived pre-B ALL cells
Consistent with these findings, our analysis of patient-derived samples showed that PTEN promoter regions are hypermethylated in B cell lymphomas (n = 68) but not in pre-B ALL samples (n = 83; Fig. 3b). Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of adult ALL, 22 cases of T cell lineage ALL and 11 cases of mature B cell lymphoma (MDACC, 1983(MDACC, -2007 23 revealed higher PTEN protein levels in pre-B ALL samples compared to T cell lineage ALL and mature B cell lymphoma (Fig. 3c). Studying a panel of normal human CD19 + sorted pre-B cells (n = 3), patient-derived pre-B ALL (n = 8) and B cell Non-Hodgkin lymphoma samples (n = 4), we confirmed these findings by Western blot (Fig. 3d). The MDACC (1983-2007) RPPA data set replicated results from previous work 24 that identified high expression levels of PTEN as predictor of favorable outcome in patients with T cell lineage ALL (Supplementary Fig. 4). In contrast, RPPA data for diagnostic samples from patients with pre-B ALL showed the opposite trend and higher than median expression levels of PTEN in diagnostic pre-B ALL samples predicted shorter relapse-free survival ( Supplementary Fig. 4).

Pten regulates AKT activity downstream of the pre-B cell receptor
To elucidate the mechanistic basis of Pten-dependency in pre-B ALL, we first tested the hypothesis that loss of Pten results in hyperactivation of PI3K-AKT signaling and subsequent cell death. In agreement with this hypothesis, we found that inducible deletion of Pten in both BCR-ABL1 and NRAS G12D -driven pre-B ALL cells resulted in increased phosphorylation of AKT at both S473 and T308 (Fig. 3e). Consistent with PTEN-dependent feedback regulation, deletion of Pten caused hyperactivation of PI3K-AKT and was followed by downregulation of pre-BCR, IL7R and CD19 (Fig. 1f-h; Fig. 3f), all of which mediate PI3K-AKT activation in pre-B cells. Syk is a B cell-specific tyrosine kinase that links pre-BCR signaling to PI3K activation [12][13][14][15] . To measure the contribution of individual components of the PI3K-AKT pathway to induce toxicity, we used selective small molecule inhibitors of the Syk (PRT06207), PI3K (BKM120) and AKT (AZD5363) kinases (Fig. 3f). Consistent with hyperactivation of AKT in pre-B ALL cells, pharmacological inhibition of AKT (AZD) largely reduced toxicity of Pten-deletion (Fig. 3g, Supplementary Fig. 5a). Also inhibition of Syk (PRT) and PI3K (BKM) mitigated the toxic effects of Pten-deletion. Given that BCR-ABL1 and NRAS G12D can activate AKT independently from Syk and PI3K, the AKT inhibitor (AZD) had the strongest protective effect in reducing cell death induced by Pten-deletion (Fig. 3h, Supplementary Fig. 5b).

Both alleles of Pten are required for survival and proliferation of pre-B ALL cells
Deletion of PTEN in human cancer typically affects one allele while the other copy is retained 25 , suggesting that cancer cells are selected for deletion of one but not both alleles of PTEN. Indeed, deletion of both alleles of Pten induces p53-dependent senescence in a model of prostate cancer, unless Tp53 is also deleted 25 . For this reason, we studied the consequences of both heterozygous and homozygous deletion of Pten in mouse pre-B ALL cells ( Supplementary Fig. 6). Comparing deletion of both alleles (Pten fl/fl ) or one (Pten +/fl ) allele of Pten, we found that-in contrast to solid tumor cells-both homozygous and heterozygous deletion of Pten affected colony formation and proliferation of pre-B ALL cells and induced cellular senescence. The deleterious effects of mono-and bi-allelic Ptenablation in pre-B ALL cells were indistinguishable ( Supplementary Fig. 6a-c). Likewise, deletion of either both or only one allele of Pten compromised leukemogenesis in an in vivo transplant experiment and no difference was noted between pre-B ALL cells carrying deletion of one or both alleles of Pten ( Supplementary Fig. 6d). Western blot analysis revealed that deletion of one allele of Pten resulted in increased phosphorylation of AKT ( Supplementary Fig. 6e). However, this increase was smaller compared to AKTphosphorylation in response to biallelic Pten-deletion.

AKT-hyperactivation eliminates autoreactive B cells
Unlike other cell types, pre-B cells are selected for an intermediate level of PI3K-AKT signaling strength [4][5][6] . Both loss-and gain-of-function mutations of PI3K trigger negative selection of pre-B cells and cell death 4-6 . PI3K-AKT signaling is activated downstream of the pre-BCR and its coreceptor CD19 [4][5] . Previous work had demonstrated that tonic activation of PI3K-AKT signaling can rescue early B cell development in the absence of a functional pre-BCR [4][5] . Here, we determined whether pharmacological inhibition of PI3K-AKT signaling can rescue negative selection and cell death in response to acute activation of an autoreactive pre-BCR. To test this hypothesis, we employed a reconstitution system 26 for inducible activation of pre-BCR signaling downstream of non-autoreactive (μ-HC NA ) or autoreactive μ heavy chains (μ-HC Auto ) or an empty vector control (EV; Fig. 4a). Tamoxifen (Tam)-induced assembly of the proximal BCR signalosome in the presence of EV or μ-HC NA had no effect on the viability of BCR-ABL1 pre-B ALL cells (Fig. 4b, Supplementary Fig. 7a). However, inducible activation of autoreactive pre-BCR signaling (μ-HC Auto ) caused rapid cell death, which could be rescued by small molecule inhibition of Syk (PRT) and AKT (AZD) kinase activity (Fig. 4b, Supplementary Fig. 7a). Activation of signaling from an autoreactive μ-HC (μ-HC Auto ) and induction of cell death involved phosphorylation of Syk and AKT, whereas activation of pre-BCR signaling downstream of μ-HC NA and EV had no effect on phosphorylation levels of Syk and AKT (Fig. 4c). Treatment with Syk (PRT) and AKT (AZD) kinase inhibitors rescued toxicity and demonstrated that hyperactivation of Syk and AKT downstream of autoreactive pre-BCR signaling was required to engage a deletional checkpoint (Fig. 4b). We conclude that hyperactivation of PI3K-AKT signaling upon deletion of Pten is functionally equivalent to hyperactivation of PI3K-AKT downstream of an autoreactive pre-BCR via Syk. Both events trigger a deletional checkpoint for removal of autoreactive B cell clones.
To demonstrate the toxic effects of Syk-and AKT-hyperactivation in a genetic experiment, we transduced pre-B ALL cells with constitutively active (CA) forms of Syk and AKT and measured pre-B ALL cell viability in the presence and absence of Syk (PRT), PI3K (BKM) and AKT (AZD) kinase inhibitors. As predicted, Syk CA rapidly induced cell death in pre-B ALL cells, which could be nearly completely rescued by kinase inhibition of Syk, PI3K and AKT (Fig. 4d, Supplementary Fig. 7b). Likewise, hyperactivation of AKT (AKT CA ) was toxic in pre-B ALL cells and toxicity was reduced by AKT kinase inhibition (AZD; Fig. 4e, Supplementary Fig. 7c). Collectively, these findings suggest that hyperactivation of PI3K-AKT signaling engages a deletional checkpoint for removal of autoreactive B cells.

B cell-specific expression and activity of PTEN
To test the basic premise of a checkpoint for elimination of autoreactive pre-B cells, we reprogrammed pre-B ALL cells into myeloid lineage cells using an inducible C/EBPα vector system 27 . To this end, we engineered Pten fl/fl pre-B ALL cells with a doxycycline inducible vector system for expression of the myeloid transcription factor C/EBPα, which results in B→myeloid lineage conversion ( Fig. 4f-h). Inducible expression of C/EBPα in pre-B ALL cells not only downregulated Pax5, a transcription factor that determines B cell lineage identity but also resulted in downregulation of PTEN levels, demonstrating that PTEN expression levels depend on B cell lineage identity (Fig. 4g). C/EBPα-mediated myeloid lineage conversion also resulted in a 45-fold increase in AKT-S 473 phosphorylation compared to the EV transduced cells, which mirrors transcriptional repression of PTEN in myeloid cells (Fig. 4g). These findings are consistent with the notion that myeloid cells, unlike B lineage cells, are permissive to loss of PTEN function and hyperactivation of AKT signaling.
To test whether B→myeloid lineage conversion erases dependency on PTEN-mediated negative regulation of AKT activity, Pten fl/fl pre-B ALL cells carrying inducible C/EBPα or empty vector controls (EV), were also induced to express Cre ( Fig. 5g-h). Inducible deletion of Pten resulted in a rapid elimination of B cell lineage ALL cells (CD19 + , Mac1 − ) as in previous experiments ( Fig. 1c-f, Supplementary Fig. 1). By contrast, deletion of Pten in B→myeloid reprogrammed (CD19 − , Mac1 + ) resulted in a slight increase in the frequency of Cre-GFP + cells, reflecting a relative survival advantage of Pten-deletion in myeloid cells (Fig. 4h). These findings are consistent with a previous study 17 , which identified PTEN as a tumor suppressor in BCR-ABL1-transformed myeloid leukemia, and reinforce the notion that normal pre-B and pre-B ALL cells are uniquely dependent on negative regulation of AKT by PTEN.

The deleterious effects of Pten-deletion in pre-B ALL are linked to B cell lineage identity
Pre-B (Ph + ALL) and chronic myeloid leukemia (CML) are both driven by the oncogenic BCR-ABL1 tyrosine kinase but markedly differ with respect to PTEN expression levels ( Fig.  5a-b). PTEN protein levels were high in BCR-ABL1-driven mouse pre-B ALL and in patient-derived Ph + ALL (n = 5) but barely detectable in mouse CML-like and patientderived CML cells (n = 5; Fig. 5a-b). This difference is consistent with regulation of Pten levels during B→myeloid reprogramming (Fig. 4g). While pre-B cells are subject to a deletional checkpoint for removal of autoreactive clones, this is not the case for other cell types (including myeloid cells). Likewise, deletion of Pten in normal myeloid progenitor cells had no effects but caused toxicity in normal pre-B cells (Fig. 5c). Moreover, Cremediated Pten-deletion had no deleterious effects in BCR-ABL1 myeloid lineage CML but caused cell death in BCR-ABL1-transformed pre-B ALL cells (Fig. 5d). Induction of cell death upon Pten-deletion in pre-B ALL cells was paralleled by accumulation of Arf, p53 and p21 checkpoint molecules. In contrast, Pten-deletion in myeloid leukemia cells neither induced activation of checkpoint molecules nor cell death (Fig. 5d-e). Consistent with its function as a regulator of an autoimmunity checkpoint, these findings suggest that PTEN is specifically required for the survival of normal and transformed pre-B cells and dispensable in myeloid cells. Activation of p53 upon acute deletion of Pten involved increased p53phosphorylation at S15, which increases p53 stability and its half-life (Fig. 5f). Interestingly, both S15-phosphorylation and accumulation of p53 were reversed by AKT-inhibition (AZD5363), indicating that activation of p53 in response to Pten-deletion is caused by AKThyperactivation (Fig. 5f).

AKT hyperactivity exacerbates glucose and energy depletion
AKT promotes glycolysis and the use of cellular ATP towards translation, cell growth and proliferation through activation of mTORC1 [28][29] . We studied the metabolic consequences of Pten-deletion on glycolysis and energy supply in the context of mTORC1 inhibition by rapamycin. As expected, inducible deletion of Pten resulted in an increase in glycolysis, as measured by increased glucose consumption and increased production of lactate ( Supplementary Fig. 8). These effects were reversed by rapamycin, indicating that increased levels of glycolysis upon Pten-deletion were caused by mTORC1 activation. Consistent with increased energy-inefficient glycolysis, inducible deletion of Pten in pre-B ALL cells reduced ATP levels, which were restored by rapamycin ( Supplementary Fig. 8). These metabolic effects of Pten-deletion were specific for B-lymphoid cells. B→myeloid reprogramming of pre-B ALL cells using inducible expression of CEBPα (Fig. 4f-h) revealed that ablation of Pten had divergent outcomes in B lymphoid and B→myeloid reprogrammed leukemia cells. While loss of Pten moderately increased glycolysis in myeloid cells, resulting in a substantial net gain of cellular ATP levels, glycolytic activity was excessively increased in B lymphoid cells resulting in depletion of glucose reserves and in near exhaustion of cellular ATP (Fig. 5g). Taken together, these findings indicate that deletion of Pten in pre-B ALL cells caused profound metabolic distress in conjunction with strong activation of p53 checkpoint activation in pre-B ALL cells. It should be noted that we tested alternative mechanisms of toxicity related to Pten deletion, including dephosphorylation of STAT5 and accumulation of cellular reactive oxygen species (ROS). Unlike metabolic stress and p53-mediated cell death, loss of STAT5-phosphorylation and increased ROS levels were only observed in BCR-ABL1-transformed ALL cells and could not be mechanistically validated in other ALL subtypes (data not shown).

Validation of PTEN as a therapeutic target in patient-derived pre-B ALL cells
Despite the well-characterized function of PTEN as a tumor suppressor, to test whether PTEN represents a therapeutic target in human pre-B ALL, we used two distinct shRNAs against PTEN and a scrambled control for lentiviral delivery in patient-derived pre-B ALL cells. The two shRNA hairpins reduced PTEN protein levels by 2-to 4-fold compared to the non-targeting (scrambled) control as determined by Western blot (Fig. 6a). Lentiviral hairpin-mediated knockdown of PTEN in three human myeloid lineage CML cell lines did not affect viability compared to scrambled controls (Fig. 6b, Supplementary Fig. 9). In contrast, PTEN knockdown induced cell death in cells from four individuals with pre-B ALL bearing BCR-ABL1 (LAX2, ICN1), TCF3-PBX1 (ICN12) or KRAS G12V (LAX7R) oncogenes ( Fig. 6b; Supplementary Fig. 9). We conclude that PTEN is essential for the survival of human pre-B ALL, validating PTEN as a potential therapeutic target in human pre-B ALL.

Preclinical evaluation of PTEN inhibitor in pre-B ALL treatment
To test potential therapeutic usefulness of pharmacological PTEN inhibition, we studied the effects of a small molecule inhibitors of PTEN, SF1670 [30][31] in human pre-B ALL cells (Fig.  6c). Biochemical validation of SF1670 activity revealed that the PTEN small molecule inhibitor induced activation of AKT signaling in pre-B ALL cases carrying BCR-ABL1 or KRAS G12V oncogenes within one hour of treatment (Fig. 6d). SF1670 strongly and selectively induced cell death in pre-B ALL cells at similar concentrations that were previously used to stimulate neutrophils in vivo 31 (IC50, 1.2 μmol/L; Fig. 6e, Supplementary   Fig. 10a). In addition, SF1670 exhibits a similar B cell lineage-specific effect as in genetic Pten-deletion and shRNA-mediated knockdown of PTEN, since myeloid CML cells were either not affected by SF1670 or only at higher concentrations (Fig. 6e). In addition, treatment of human pre-B ALL cells with SF1670 phenocopied the effects of genetic deletion of Pten fl/fl in pre-B ALL cells. Importantly, acute deletion of Pten in Pten fl/fl in pre-B ALL cells resulted in phosphorylation (S15) and activation of p53 (Fig. 5f). Likewise, small molecule inhibition of PTEN resulted in rapid activation of p53 and p21 checkpoint molecules and p53-phosphorylation at S15 (Fig. 6f). To test whether p53-checkpoint activation represents an important aspect of toxicity in pre-B ALL cells resulting from acute PTEN-inhibition, we transduced Tp53 fl/fl BCR-ABL1 transformed pre-B cells with Cre or empty vector (EV). Interestingly, Tp53 deletion caused a substantial shift in dose response curves of cells treated with SF1670. (Fig. 6g). While Cre-mediated deletion of Pten resulted in rapid loss of CD19 and pre-BCR (VpreB) surface expression (Fig. 1f, Supplementary Fig.  10b), we observed the same effects in human pre-B ALL cells that were treated for two days with SF1670 ( Supplementary Fig. 10b). Because prolonged systemic inhibition of PTEN would likely raise safety concerns based on its role as tumor suppressor in a wide range of cell types 8,18 , we tested the effects of short, transient inhibition of PTEN using SF1670. One single exposure to SF1670 for three hours (and subsequent washout of SF1670) induced AKT hyperactivation and caused significant cell death in pre-B ALL cells (P < 0.001; Fig.  6h).
Small molecule inhibition of PTEN (SF1670) also recapitulated metabolic features of genetic deletion of Pten (Fig. 6i). While SF1670-treatment resulted in moderate stimulation of glycolysis (glucose consumption and lactate production) in CML cells, stimulation of glycolytic responses were much stronger in B lymphoid ALL cells (Fig. 5g). However, SF1670-treatment resulted in a net increase in cellular ATP in myeloid CML cells but in near-complete depletion of energy reserves in patient-derived pre-B ALL cells. Recapitulating our measurements in response to Cre-mediated deletion of Pten in myeloid leukemia and pre-B ALL (Fig. 5g), SF1670-treatment induced pre-B cell-specific depletion of ATP and energy crisis.

Discussion
Unlike other cell types, pre-B cells are under intense selective pressure and have to pass a central B cell tolerance checkpoint for removal of autoreactive clones 2 . The vast majority of newly generated pre-B cells express an autoreactive immunoglobulin μ-heavy chain resulting in strong pre-B cell receptor signaling and are, hence, eliminated at this checkpoint 2-3 . Humans carrying germline mutations resulting in hyperactive PI3K-AKT signaling suffer from profound B cell lymphopenia 6 , presumably because virtually all newly generated pre-B cells are subject to negative selection due to hyperactive PI3K-AKT signaling. Recent work from our group demonstrated that central B cell tolerance checkpoints are fully functional in human pre-B ALL cells, despite malignant transformation 31,32 . Here we demonstrated that PTEN represents a critical gatekeeper of this checkpoint. Despite its tumor suppressor function in all major subtypes of cancer, PTEN is required to prevent indiscriminate checkpoint activation. Reflecting the unique function of PTEN in central B cell tolerance, pre-B ALL was the only subtype of human cancer that we found exempt from PTEN lesions (Fig. 3a). Previous work demonstrated that pre-B cells need a minimum level of PI3K-AKT signaling to survive 5 and targeting of this pathway is potentially useful in the treatment of human pre-B ALL 33 . However, here we propose that acute inhibition of PTEN or direct pharmacological hyperactivation of PI3K-AKT signaling may represent a strategy to trigger central B cell tolerance checkpoints and thereby overcome conventional drug resistance in human pre-B ALL.

Patient samples, human cells and cell lines
We obtained primary patient samples (Supplementary Table 1) in compliance with the Institutional Review Board of the University of California San Francisco. Human leukemia cells were maintained in Minimum Essential Medium α (MEM α, Invitrogen, Carlsbad, CA) supplemented with GlutaMAX, 20% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and incubated at 37°C in a humidified incubator with 5% CO 2 . We obtained informed consent from all participants. Primary human ALL cells (patient samples) were cultured on irradiated OP9 stromal cells (ATCC; Manassas, Virginia) in same media as described above. Table 2. Transfections of the MSCV-based retroviral constructs were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with Opti-MEM media (Invitrogen). Retroviral supernatant was produced by co-transfecting 293FT cells with the plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env). Cultivation was performed in high glucose Dulbecco's modified Eagle's medium (DMEM, Invitrogen) with GlutaMAX containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 25 mM HEPES, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids. Regular media were replaced after 16 hours by growth media containing 10 mM sodium butyrate. After 8 h incubation, the media was changed back to regular growth media. 24 hours later, the virus supernatant were harvested, filtered through a 0.45 μm filter and loaded by centrifugation (2000 × g, 90 min at 32°C) two times on 50 μg/mL RetroNectin (Takara, Madison, WI) coated non-tissue 6-well plates. Three million cells were transduced per well by centrifugation at 600 × g for 30 minutes and maintained for 72 h at 37°C with 5% CO 2 before transferring into culture flasks.

Preparation of bone marrow cells from mice
Complete list of mice in this study is provided in Supplementary Table 3. Bone marrow cells were extracted from mice younger than 6 weeks of age. Bone marrow cells were obtained by flushing cavities of femur and tibia with PBS. After filtration through a 70 μm filter and depletion of erythrocytes using a lysis buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage (in 10% DMSO, 90% FBS) or subjected to further experiments.

BCR-ABL1 -driven mouse model of pre-B ALL and myeloid leukemia
Bone marrow cells from Pten fl/fl (n = 3, female) and Pten fl/+ (n = 2, female) mice (Supplementary Table 3) were collected and retrovirally transformed with BCR-ABL1. For B cell lineage leukemia, transduction was performed after culturing of bone marrow cells with 10 ng IL-7/ml for one week. To generate myeloid lineage leukemia bone marrow cells were cultured with 10 ng/ml recombinant mouse IL-3, 25 ng/ml recombinant mouse IL-6, and 50 ng/ml recombinant mouse SCF for one week (PeproTech). This treatment leads to selection for Lin − Sca-1 + c-Kit + (LSK) cells. Transformation of precursor B cells and LSK cells with BCR-ABL1 generate pre-B ALL and CML-like models respectively. After BCR-ABL1 transduction all cytokines were removed from the culture to negatively select the nontransduced cells. Both ALL and CML cells were maintained in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with GlutaMAX, 20% fetal bovine serum, 100 IU ml −1 penicillin, 100 μg ml −1 streptomycin and 50 μM 2-mercaptoethanol. BCR-ABL1-transformed ALL cells were propagated only for short periods of time and usually not longer than 2 months to avoid acquisition of additional genetic lesions during long-term cell culture.

In vivo transplantation of leukemia cells
For in vivo experiments, mouse ALL cells were labeled with retroviral firefly luciferase, selected based on antibiotic resistance (Blasticidin), and injected via tail into sublethally irradiated (300 cGy) NOD/SCID recipient mice. Engraftment was monitored using luciferase bioimaging (VIS 100 bioluminescence/optical imaging system; Xenogen). D-Luciferin (Xenogen) dissolved in PBS was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the light emission. All mouse experiments were subject to institutional approval by the University of California San Francisco Institutional Animal Care and Use Committee. 6-8 week old female NOD-SCID mice (Jackson Laboratories, ME) were randomly allocated into each treatment group. The minimal number of mice in each group was calculated by using the 'cpower' function in R/Hmisc package. No blinding was used.

C/EBPα-mediated reprogramming of pre-B cells into myeloid cells
The TetO-C/EBPα-GFP-construct was generated as a modification of the previously published TetO-CEBPα-tdTomato lentivirus 27 . Pten fl/fl pre-B ALL cells were first transduced with rtTA, selected for puromycin resistance and subsequently infected with the TetO-C/EBPα-GFP-construct. C/EBPα was induced in the Pten fl/fl pre-B ALL cells by addition of 2 μg/ml doxycycline (Sigma-Aldrich). Staining for cell surface antigens was done with directly conjugated antibodies against CD11b (PE; Mac1) and CD19 (PerCP-Cy5.5) EBioscience. To measure the effects of Pten-deletion in C/EBPα-reprogrammed pre-B ALL cells, deletion was induced with a tamoxifen-inducible Cre or an empty vector control (0.5 μmol/L). DAPI and 7AAD at 1 μg/ml were used as viability markers.

Array-based promoter methylation analysis using HELP
The HELP (HpaII tiny fragment enrichment by ligation mediated PCR) assay was performed as previously described. One microgram of high molecular weight DNA was digested overnight with isoschizomer enzymes HpaII and MspI respectively (NEB, Ipswich, MA). DNA fragments were purified using phenol/chloroform, resuspended in 10mM Tris-HCl pH 8.0, and used immediately to set up the ligation reaction with MspI/HpaII-compatible adapters and T4 DNA ligase. Ligation-mediated PCR was performed with enrichment for the 200 to 2,000 bp products and then submitted for labeling and hybridization onto a human HG_17 promoter custom-designed oligonucleotide array covering 25,626 HpaII amplifiable fragments within the promoters of the genes. To analysis, raw data (.pair) files were generated using NimbleScan software. Signal intensities at each HpaII amplifiable fragment were calculated as a robust (25% trimmed) mean of their component probe-level signal intensities. Any fragments found within the level of background MspI signal intensity, measured as 2.5 mean-absolute-deviation (MAD) above the median of random probe signals, were considered as "failed" probes and removed. A median normalization was performed on each array by subtracting the median log-ratio (HpaII/MspI) of that array (resulting in median log-ratio of 0 for each array).

Western blotting
Cells were lysed in CelLytic buffer (Sigma, St. Louis, MO) supplemented with PMSF, phosphatase and protease inhibitor cocktail (Pierce, Rockford, IL). 15 μg of protein mixture per sample were separated on NuPAGE (Invitrogen, Carlsbad, CA) 4-12% Bis-Tris gradient gels and transferred on PVDF membranes (Immobilion, Millipore, Temecula, CA). For the detection of mouse and human proteins by Western blot, primary antibodies were used together with the WesternBreeze immunodetection system (Invitrogen). Details of antibodies used are in Supplementary Table 4.

Flow cytometry
Antibodies used for flow cytometry experiments are listed in Supplementary Table 4. For apoptosis analyses, Annexin V, propidium iodide and 7-AAD (BD Biosciences) were used. Fortessa scanner was used for flow cytometry and FlowJo-V10 was used for data analysis.

Senescence-associated β-galactosidase assay
Senescence-associated β-galactosidase activity was performed on cytospin preparations. Briefly, a fixative solution (0.25% glutaraldehyde, 2% paraformaldehyde in PBS pH 5.5 for mouse cells) was prepared fresh. 1 g paraformaldehyde was dissolved in 50 ml PBS at pH 5.5 by heating followed by addition of 250 μl of a 50% stock glutaraldehyde solution. 1× Xgal staining solution (10 ml) was prepared as follows: 9.3 ml PBS/MgCl 2 , 0.5 ml 20× KC solution (that is, 820 mg K 3 Fe(CN)6 and 1,050 mg K 4 Fe(CN)6 × 3H 2 O in 25 ml PBS) and 0.25 ml 40× X-gal (that is, 40 mg 5-bromo-4-chloro-3-indolyl β-D-galactoside per milliliter of N,N-dimethylformamide) solutions were mixed. 200,000 cells per cytospin were used (700 rpm, 8 min). The fixative solution was pipetted onto cytospins and incubated for 10 min at room temperature, then washed twice for 5 min in PBS/MgCl 2 . Cytospin preparations were submerged in 1× X-gal solution, incubated overnight at 37 °C in a humidified chamber and washed twice in PBS. Images were acquired with a regular microscopy and percentage of stained cells calculated by counting.

Colony forming assay
Methylcellulose colony-forming assays were performed with 10,000 cells after transformation with Cre-ER T2 or ER T2 and 2 days of treatment with 0.5 μmol/l tamoxifen. Cells were resuspended in murine MethoCult medium (StemCell Technologies, Vancouver, BC, Canada) and cultured on dishes (3 cm in diameter) with an extra water supply dish to prevent evaporation. After 7 to 14 days, total numbers of colonies per plate were counted by microscopy.

Cell-cycle analysis
For cell-cycle analysis, the BrdU flow cytometry kit for cell-cycle analysis (BD Biosciences) was used according to manufacturer's instructions. BrdU incorporation (APC-labelled anti-BrdU antibodies) was measured along with DNA content (7-amino-actinomycin-D) in fixed and permeabilized cells. The analysis was gated on viable cells that were identified based on scatter morphology.

Cell viability assay
100,000 cells per well were seeded in a volume of 100 μl medium on Optilux 96-well plate (BD Biosciences, San Jose, CA). Inhibitors were diluted in medium and added at the indicated concentration in a total culture volume of 120 μl. After 3 days in culture, 12 μl of Resazurin (R&D, Minneapolis, MN) was added to each well and incubated for 4 hours at 37 °C. The fluorescent signal was monitored using 535 nm excitation wavelength and 590 nm emission wavelength. Fold changes were calculated using baseline values of untreated cells as a reference (set to 100 percent). Each sample was measured in triplicates.

RNA purification and Expression analysis
Total RNA was purified using the RNeasy kit (Qiagen, Valencia, CA). RNA quality was checked by using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA was generated from 5 μg of total RNA using a poly(dT) oligonucleotide and the SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). Biotinylated cRNA was generated and fragmented according to the Affymetrix protocol and hybridized to mouse Gene 1.0 ST (Affymetrix, High Wycombe, UK). After scanning (GeneChip Scanner 3000 7G; Affymetrix) of the GeneChip arrays, the generated CEL files were imported to BRB Array Tool (http://linus.nci.nih.gov/BRB-ArrayTools.html) and processed using the RMA algorithm (Robust Multi-array Average) for normalization and summarization. The GEO accession number for the arrays is GSE34829.

Proteomic profiling
Proteomic profiling was performed using Reverse Phase Protein Array (RPPA) analysis 23 on peripheral blood and bone marrow specimens from 192 patients with acute lymphocytic leukemia (ALL) including 192 samples at diagnosis and 12 paired diagnosis-relapse samples evaluated at The University of Texas M.D. Anderson Cancer Center (MDACC) between 1983 and 2007 (two from the 1980s, 45 from the 1990s). Samples were acquired during routine diagnostic assessments in accordance with the regulations and protocols (Lab 01-473 after September 2001, prior protocols for samples before then) approved by the Investigational Review Board (IRB) of MDACC. Informed consent was obtained in accordance with Declaration of Helinski. Samples were analyzed under and IRB-approved laboratory protocol (Lab 05-0654). Samples were selected for inclusion based on availability in the Department of Leukemia Sample Bank. Patients aged 15 or over were included; another 8 pediatric ALL cases were on the array but are not included in this analysis. Samples underwent Ficoll separation to yield a mononuclear fraction. The samples were normalized to a concentration of 1 × 10 4 cells/μL and a whole cell lysate prepared as previously described 28 . All protein samples were prepared from fresh cells on the day of collection. Of the 192 newly diagnosed ALL cases most were treated with the hyper CVAD regimen either alone (n = 91) or in combination with rituximab (n = 31) or a tyrosine kinase inhibitor (n = 28) or nelarabine (n =3). A modification of the Berlin-Frankfurt-Mannheim regimen was used in 23 cases. The remaining 16 received miscellaneous combination regimens in use over this time period.

Glucose, Lactate and ATP Measurements
Glucose and lactate levels were measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen) and the L-Lactate Assay Kit (Cayman Chemical), respectively, according to the manufacturers' protocols. Glucose and lactate concentrations were measured in fresh and spent medium. Total ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer's protocol. 10 6 cells per mL were seeded in fresh medium and treated as indicated in figure legends. Relative levels of glucose consumed, lactate produced and total ATP are shown, and all values were normalized to number of viable cells. Inhibitors used in this study are listed in Supplementary Table 5.

Reconstitution model for pre-BCR signaling
Pre-B cells from Blnk −/− Rag2 −/− Igll1 −/− triple knockout transgenic mice 26 were engineered to express either a non-auto-reactive (μ-HC NA ), an autoreactive pre-BCR (μ-HC Auto ) or an empty vector control (EV). The cells were transduced with a tamoxifen-inducible (Blnk-ER) form of the pre-BCR linker molecule Blnk (also known as SLP-65). Addition of tamoxifen (Tam) releases Blnk from its complex with HSP90 and allows for assembly of the pre-BCR signaling complex within minutes.

Statistical analysis
Unpaired, two-tailed Student's t test was used to compare colony number, S phase percentage and frequency of cellular senescence between different groups. Two-sided Mann-Whitney Wilcoxon test was used to compare methylation values of ALL vs. normal pre-B or B-cell lymphoma groups and RPPA data of newly diagnosed adult ALL vs. T cell lineage ALL or mature B cell lymphoma , using R version 2.14.0. Mantel-Cox log rank test (twosided) was used to compare survival data between different groups. R package "survival" version 2.35-8 was used for the survival analysis.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  transformed pre-B ALL cells transduced with Cre-ER T2 or empty vector (EV) control (right). In the latter experiment all mice were treated with Tamoxifen (0.4 mg/mouse; n = 7 per group). P values were calculated by Mantel-Cox log-rank test. All cells were labeled with luciferase before injection. Treatments were started on day 5 after transplantation and continued for ten consecutive days. Error bars (a-b, d) represent S.D.   (d) Western blot measuring the activity AKT signaling pathway after treatment of two pre-B ALL cases with SF1670 (10 μmol/L). β-actin was used as loading control. Representative blot, n = 4. (e) Cell viability assay after SF1670 treatment of human pre-B ALL (n = 5, red) and CML (n = 3, green) cases. P < 0.0001 was calculated using contingency table comparing Pre-B ALL vs CML cells. Error bars represent SD. (f) Western blot measurement of p21, p53 and p53 (Ser15) on a pre-B ALL cells treated with SF1670 (10 μmol/L). β-actin was used as loading control. Representative blot, n = 3. (g) Cell viability assay after SF1670 treatment of BCR-ABL1transformed pre-B ALL cells with or without Tp53 deletion. Error bars represent SD. P < 0.001 was calculated by contingency table. (h) Measuring viability of two pre-B ALL cases treated for only 1 or 3 h with SF1670 (10 μmol/L) for three consecutive days. P values were calculated by contingency table comparing average cell viability after each treatment with vehicle treated cells. The results are representative of three independent experiments. (i) Measuring Glucose consumption, lactate production, and ATP levels were after 24 hours treatment of CML cell line (KYO-1) and patient derived pre-B ALL case (LAX7R) with SF1670 (2.5 μmol/L). Values obtained were normalized to number of viable cells and are shown as average relative levels. P values were calculated by Student's t test. Error bars represent SD.


A Novel Shiga Toxin 1a-Converting Bacteriophage of Shigella sonnei With Close Relationship to Shiga Toxin 2-Converting Pages of Escherichia coli

In recent studies, strains of non-dysenteriae 1 Shigella (NDS) expressing Shiga toxin have been reported. In this study, we report a novel stx1a-converting bacteriophage of Shigella sonnei associated with travel to Mexico. Phylogenetic comparison between this and other stx-converting phages suggests that toxigenic NDS strains have arisen through separate horizontal transfer events from toxigenic Escherichia coli.

Previously, the expression of Stx toxins in Shigella species was believed to be unique to Shigella dysenteriae type 1. More recently, stx-encoding isolates of Shigella sonnei [4][5][6], S dysenteriae type 4 [7], and Shigella flexneri [8] have been reported. These isolates were associated with travel to Eastern Europe [4,5], Morocco [6], and Hispaniola [7,8]. The details of stx acquisition by these non-dysenteriae 1 Shigella (NDS) isolates remain unclear, including the chronology of toxin acquisition (ie, recent vs remote), the origin of the converting phages, and their global distribution.

METHODS
Stool samples were screened for Stx by immunoassay according to the manufacturer's instructions (ImmunoSTAT! EHEC assay; Meridian Biosciences, Cincinnati, OH). Stool cultures were performed using Hektoen enteric and XLD selective agars, and automated biochemical speciation of suspicious colonies was performed (Vitek2, Durham, NC). A toxin-negative clinical isolate of S sonnei was isolated by similar means and used as a control. Identifications were confirmed by the San Diego County Public Health Laboratory.
Phage lysis was induced by treating Shigella cultures with 0.5 µg/mL mitomycin C (MMC) as previously described [8]. After MMC addition, culture aliquots were removed hourly for optical density (OD 600 ) reading. Large-scale phage genomic DNA isolation was performed after the QIAGEN Lambda kit protocol (QIAGEN, Valencia, CA).
Restriction analysis was performed by digesting purified phage genomic DNA with EcoRI and HindIII according to the manufacturer's recommended conditions (New England BioLabs, Ipswich, MA), and restriction products were resolved on 0.7% agarose. Invitrogen 1 kb plus ladder was used as a size standard (Invitrogen, Carlsbad, CA).
Sequencing library preparation was performed from genomic bacterial DNA and purified phage genomic DNA. Deoxyribonucleic acid was fragmented to 350 base pairs (bp) using the Covaris M220 (Covaris, Woburn, MA). The Illumina TruSeq Nano Kit (Illumina, San Diego, CA) was used to create barcoded libraries according to the manufacturer's protocol. Equimolar amounts of each library were pooled, and bidirectional sequencing was performed using the Illumina MiSeq platform for 300 cycles, using v2 reagents. The phage genomic sequence was determined by de novo assembly using the SPAdes 3.5.0 algorithm. To identify the integration site, bacterial genomic sequencing reads containing the phage terminal sequences were extracted, aligned, and subjected to Basic Local Alignment Search Tool (BLAST) search. The complete phage genome sequence is available through GenBank under accession number KR781488.
For phylogenetic analysis, phage sequences were obtained from National Center for Biotechnology Information using accession numbers found in Supplementary Table 1. Multiple sequence alignment was performed using MAFFT version 7, and maximum likelihood trees were inferred using RAxML v7.0.3 with general time reversible + gamma nucleotide substitution model [9]. Branch support was performed using 100 bootstrap iterations; all branches had bootstrap scores of >90, except the HUN/2013 -Stx1phi branch, which had a score of 58.

RESULTS
The stx1a-converting phage described here was isolated from S sonnei that infected 2 epidemiologically unrelated subjects who presented to our hospital in 2014. Both subjects had recently returned from Baja California Norte, Mexico, and presented with diarrhea and abdominal discomfort. Both were treated with intravenous fluids and oral fluoroquinolone antibiotics, and both made uneventful recoveries. Stool samples from both individuals were positive for Stx by immunoassay, and, unexpectedly, stool culture on selective agar grew S sonnei from both subjects. Shigella sonnei species was confirmed by automated biochemical species determination and confirmed by polymerase chain reaction targeting a methylase gene sequence that is unique to S sonnei [10] (data not shown). The Stx-producing isolates were named Ss-VASD01 and Ss-VASD02.
Treatment of S sonnei cultures with the DNA-alkylating agent MMC induced lysis of both Stx-expressing isolates but not of an Stx-negative S sonnei isolate ( Figure 1A), suggesting the presence of an inducible bacteriophage. Phage particles were purified from MMC-induced cultures by polyethylene glycol precipitation, and restriction analysis of purified phage genomic DNA revealed a large molecular weight genome from both isolates, which shared identical restriction patterns ( Figure 1B).
To further characterize the phage (named ɸSs-VASD), deep sequencing of purified phage and the whole bacterial genome was performed. De novo assembly of sequencing reads resulted in a 62 851-bp genome with 712.5× mean coverage. A total of 189 sequencing reads were recovered that spanned the host-phage junction, and BLAST alignment of the non-phage portions revealed 100% identity to the wrbA gene of S sonnei reference strain Ss046 (host-phage junction at Ss046 position 1088102).
To assess the relationship of ɸSs-VASD to other stx-converting phages, we performed phylogenetic comparisons between the genomic sequences of ɸSs-VASD and 18 previously reported stx-converting phages (Supplementary Table 1  phage genome sequences were noted to cluster by integration site preference, with distinct clusters containing wrbA-integrating isolates ( Figure 1C). The closest genetic relationships were found between ɸSs-VASD and stx2-converting phages of E coli.
Analysis of the stx coding sequence of ɸSs-VASD revealed 100% amino acid identity to 7/8 stx1-converting phages, with only POC-J13 varying from the consensus sequence at 2 positions (P 284 Q in stxA and A 11 T in stxB). ɸSs-VASD was found to contain the stx operon promoter P R' , which drives fluoroquinolonemediated toxin production from stx2-encoding phage and contributes to toxin expression in stx1-encoding phages [3,11,12].

DISCUSSION
In this study, we report a novel stx1a-converting phage of S sonnei associated with travel to Baja California, Mexico. Prior reports of Stx-expressing NDS have been associated with travel (Hispaniola for S dysenteriae serotype 4 and S flexneri, and to the Eastern Europe and Morocco for S sonnei). To our knowledge, these are the first such isolates originating from North America. Furthermore, it appears that the subjects in this study may be part of a larger outbreak of Stx-toxigenic S sonnei, presently under investigation by the California Department of Public Health [13]. These findings suggest an expanding global emergence of Stx-toxigenic NDS. Alternatively, the presence of Stx-toxigenic phage in NDS may have been overlooked because, until recently, it was not common practice to screen diarrheal stools for Shiga-like toxins.
One concern surrounding Shiga toxin-producing bacterial strains is the potential for toxin induction by antibiotics. Fluoroquinolone antibiotics are of particular concern, because they can induce toxin production by inducing bacterial DNA damage responses. Even though ɸSs-VASD is inducible by DNA damage and encodes a fully intact stx operon, treatment with fluoroquinolone antibiotics in both subjects reported here did not appear to trigger any clinical worsening or complications. In fact, to our knowledge, there have been no cases of complications associated with Stx-NDS infection, an observation that could be explained by several theories including the predominance of the less-pathogenic Stx1 variant [2] or variability in toxin induction by antibiotics. Alternatively, there may simply be an insufficient numbers of cases detected thus far (even in outbreaks involving high-risk STEC isolates, HUS occurs in the minority of cases).
It is interesting to note that phylogenetic analysis of ɸSs-VASD reveals a relationship to other wrbA-integrating phages. This relationship seems to be irrespective of stx type, because the closest relationship was to ɸ191 and p13374, 2 stx2-converting phages of STEC associated with outbreaks of enteroaggregative hemorrhagic E coli. Given the overall similarity of these phages apart from the stx operon, the findings implicate a recombination event leading to similar phages with differing toxin operons, a phenomenon that has been observed in STEC phages [14].
A weaker phylogenetic relationship was observed between ɸSs-VASD and the S flexneri phage POC-J13, and the S sonneiconverting phage 75-02.
The origin of stx-converting phages in NDS stains is uncertain, as is the chronology of their existence. Possible theories include (1) recent horizontal transfer of phage from STEC and (2) longstanding presence of stx-converting phages in NDS that was previously undetected. The high degree of genetic similarity between ɸSs-VASD and previously reported stxconverting phages of STEC supports the theory that Ss-VASD arose from horizontal transfer between STEC and NDS strains, rather than by parallel evolution within NDS. We are not able to perform precise molecular clock analysis; however, the absence of major sequence divergence between ɸSs-VASD and STEC phages is suggestive of a recent transfer event. The occurrence of horizontal transfer events such as this seems plausible, given the discovery of large amounts of stx-converting phage found in human and agricultural wastewater [15].

CONCLUSIONS
Important future directions will include characterization of additional Stx-NDS phages, which will create better understanding of their diversity and may allow more precise inference of their origins. These findings suggest the need for more careful clinical and epidemiological monitoring for Shiga toxigenic S sonnei, because these strains seem to be broadly distributed globally, and infections therewith have the potential to cause complications not previously associated with S sonnei.


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4. Detection of Toxoplasma Gondii Infection by Polymerase Chain Reaction (PCR) and Histological Examination on Balb/c Mice

The purpose of this research was to compare the use of PCR method and histological examination to diagnose toxoplasmosis in tissues of Balb/c mice infected with sporulated oocysts through drinking water. A total of 20 male Balb/c mice aged approximately 2 months were used in this experiment. Each mouse was infected with 1x103 Toxoplasma gondii tachyzoites intraperitoneally. Tissue samples (liver, lung, heart, kidney, and brain) were collected from 5 mice on day 1, day 5, day 7, and day 9 after infection. Samples were then examined by PCR and histological methods. The data collected were analyzed descriptively. The results showed that PCR method was more sensitive than histological examination. PCR examination using primer invitrogen gen can amplify DNA T. gondii at 436 bp of the samples from liver, lung, heart and brain on Day 7 and Day 9 after infection. The histological examination showed that the cyst of toxoplasma was found in the brain while mononuclear cells infiltration was found in other internal organs.

Background
Toxoplasma gondii is a worldwide parasite that can infect the central nervous system of warm-blooded animals, including humans (Montoya and Liesenfeld, 2004). Domestic cats and other felids have been shown as the major reservoir hosts of this parasite. It is generally assumed that cats play a major role in transmitting T. gondii through faecal contaminated soils, foods or water because they excrete millions of oocysts in a short period of time (1-2 weeks) (Dubey, 2008).
Toxoplasmosis caused by T. gondii infection has become a major public health concern recently due to the immunosuppressive effect of the ravaging HIV/AIDS pandemic (Lindstrom et al., 2006;Uneke et al., 2007). Several laboratory methods have been developed to detect antibody in the serum of infected cats such as PCR, ELISA, LAT (latex agglutination test), IHA (indirect hemagglutination assay), IFAT (indirect fluorescent antibody test) and immunochromatography (IC). Though these tests are more sensitive and specific, they are expensive and require a specialized Open Acces 21 laboratory (Zhang et al., 2009). IC is rapid, simple, sensitive, and specific. It will be a suitable diagnostic tool for detection of the specific antibodies in T. gondii infection in cats under field conditions (Xiaohong et al., 2004).
Bioassay in mice, although specific and relatively sensitive for the detection of T. gondii oocysts, relies on the presence of sporulated ocytests and thus infective oocysts in samples. Not only does this method introduces a possible biohazard element, it is also expensive, requires animal facilities, and is time consuming (Salant et al., 2007).
Faecal flotation technique is used to detect oocyst presence in faecal samples as well. Although flotation is a reference method for the detection of T. gondii oocysts, it has been suggested that an alternative test is also needed because microscopic examination is time consuming and needs an experienced microscopist (Greene, 2006).
In a routine histological examination, which is stained with hematoxylin and eosin (H&E), T. gondii antigen is generally invisible, especially when the number of the antigen is limited or dissolved (Lappin, 1994). Diagnosis using PCR from tissue samples (placenta, brain, liver and lung) has been conducted in many countries and shown satisfying result. Therefore, it can be assumed that PCR method has higher sensitivity and specificity to diagnose toxoplasmosis infection compared with conventional methods (Bell and Ranford-Cartwright, 2002;Contini et al., 2005;Calderaro et al., 2006;Bastien et al., 2007).
According to Edvinsson et al. (2006) PCR method can detect parasites in small amount or even only one with high specificity from various tissues, sputum, cerebrospinal fluid, blood, urine, and faeces. Based on the above explanations, this research was intended to study further the use of PCR method and histological examination on various tissue sampless (liver, lung, heart, kidney, and brain) for toxoplasmosis diagnosis using Balb/c mice infected with sporulated oocysts through drinking water as experimental animals

Materials and Methods
This research used 20 Balb/c mice. Oocysts were collected from faeces whereas T. gondii tachyzoites were obtained from some internal organs of the mice such as liver, lung, heart, and brain.

Toxoplasma gondii Oocysts Isolation from Cat's Faeces
Isolation of T. gondii from faeces was done follows Lappin (1994). In brief, faeces was floated in a container, stored at 4  C and treated with water for 2 days till softened. Sulphuric acid 2% was added to change faecal emulsion into paste. The faeces was then resuspended in a 10-time volume of Sheather sugar solution (40% of sucrose in 0,8% of phenol as the preservatives). Suspension was filtered with 710 µm sieve (Endecott Ltd. England) to remove big particles. The filtrate was centrifuged at 3000 rpm for 10 minutes. Supernatant, 2 ml, was drawn using a pasteur pipette, inserted into a bottle and added with potassium bichromate 2%. The solution was stored in a sealed bottle at room temperature for 3-7 days so that the oocysts sporulated. The mixture was filtered through a 44 µm sieve. The remaining parts were sprayed with distilled water and then used for toxoplasmosis examination. Sporulated oocysts were given to 20 mice through drinking water.

Isolation from Mouse Peritoneal Fluid
All infected mice, as marked by the formation of ascites liquid, were sacrified. Their stomaches were cleansed three times using 5 ml of physiological NaCl solution to obtain tachyzoites. Tachyzoites was used to infect 10 adult mice with dilution dosage of 1 X 10 7 to obtain more tachyzoite using a technique similar to the previous ones. The cleansed stomach was centrifuged at 3000 rpm (Beckman), 4°C for 10 minutes. Pellets were washed 3 times using PBS solution pH 7.4 which contained tris-ammonium-chloride to lyse red blood cells contaminants. The exudate was incubated by being shaken at 37 C for 5 minutes to lyse red blood cells. The suspension was added with 40 ml of PBS, carefully stirred, and centrifuged at 3000 rpm for 10 minutes. These cleansing processes were repeated at least 4 times to eliminate unwanted proteins. After the final centrifugation process, cells were resuspended in 100 ml of PBS. Using a 27size needle, the suspension was drawn carefully so that the pellets containing peritoneal cells were not included. Free trophozoites suspension was then filtered with a 3 µm milipore sieve to remove cell debris (Salant et al, 2007).

Sample Collection
Tissue samples (liver, lung, heart, and brain) were collected from each of 5 mice on Day 1, 5, 7, and 9 after the infection for further process. These samples were used for DNA isolation and histological preparations.

Isolating DNA from tissues
A part of tissue samples (liver, lung, heart, kidney and brain) was used as histological preparation while the other part was scaled and grained in a mortar. These samples were then treated with liquid nitrogen and pounded till homogenized. Next, the sample was added with buffer lysis (b) with the ratio of 1 sample: 9 buffer lysis (b). Then the mixture was incubated at temperature of 37 0 C for 15 minutes. After the incubation, the sample was centrifuged at the speed of 12.000 rpm for 15 minutes, and the supernatant was washed 3 times with sterile PBS with 7,2 pH. The pellet was added with buffer of digestion with the ratio of 500 mg sample: 1 ml buffer of digestion. The sample was then incubated in water bath at the temperature of 55 o C for 2 hours and boiled at the temperature of 100 o C for 8 minutes. It was then centrifuged at 12,000 rpm speed for 30 seconds. After that, the supernatant was taken to be amplified by PCR (Matsuura et al, 1990).

Amplifying DNA with PCR
Amplification of DNA samples was done by using PCR (Ready To Go TM PCR Beads from Amersham Pharmacia Biotech) commercial kit. The primers used, name 5  -GAC TCG GGC CCA GCT GCG -3  (forward) and 3  -CCT CTC CTA CCC CTC CTC -3  (backward), were purchased Invitrogen, Berlin Buch GmbH Jerman. PCR reaction mixtures were prepared ifollwing the protocol provided. Here, 5 µl of DNA targets, 2 l of forward and reverse primers (final concentration of 2 pmol), and sterile aquadest up to 25 µl were added to a PCR tube containing a PCR bead. PCR reaction was done in the following condition: one cycle of preparation stage at 95  C for 5 minutes, followed by 40 cycles for denaturation at 94 C for 1 minute, annealing at 60 o C for 30 seconds, elongation at 72  C for 1 minute and termination at 72  C for 10 minutes. PCR results were visualized using 1% of agarose electrophoresis; 0.5 x TBE (Tris borate buffer) using cDNA as the positive control.

Histological preparation
Tissue samples were poured in formalin 10%, cut into small pieces, inserted into formalin 4%, and then continued with dehydration process with graded alcohol (ethanol 80%, 95%, 100%), each for 2 hours. The next step was purification with xylol, blocked with paraffin, and cut using microtome. The cut tissues was mounted onto the surface of abject glasses, fixated and stained with haematoxylin-eosin (HE) staining. The occurrence of the parasite (in the form of bradyzoites cyst) inside the tissue was confirmed by miscroscopic examination using 100x Phase contrast, oil immersion objective lens with an Olympus binocular microscope.

Data analysis
Results obtained from PCR and histological examinations were analyzed descriptively.

Results
The result of 20 mice infected with T. gondii oocyst through drinking water can be seen in Figure 1.

Isolation and Amplification of DNA from the Mice Organ using PCR
The result of PCR examination on organs of Balb/c mice to diagnose the toxoplamosis through infection by providing oocysts which had been sporulated can be seen in Table 1. The result of examination on DNA in the organ of Balb/c mice which toxoplasma DNA was succesfully isolated by using primer invitrogen shows that the DNA is 436 bp. It can be seen in Figure 2.

Histological Preparation
Histological examinations successfully found one cyst in the brain of infected mice as can be seen in Figure 3. This finding was somewhat different from what previously obtained by Hanafiah et al. (2013) in a research applying scouring method that a cysts was found in both brain and hearth Figure 3. A cyst found in the brain tissue of the mouse containing bradyzoites with HE coloring (zoomed at 10 x 40) As showed in Figure 4 in other organs, which were free from cysts, only infiltrated mononuclear cells were found. Results of PCR and histological methods performed to particularly detect the occurance of 436 bp DNA sequence and bradyzoite cysts in the liver, lung, heart, kidney and brain of infected mice can be seen in Table 2.

Discussion
The result of PCR examinations found T. gondii DNAs detected in liver, lung, heart, and brain samples collected on day 7 and 9 after infection, but not from those collected on day 1 and 5 (Table 1). These might be caused by: until day-5 after infection the parasite had not yet grown or the amount of DNA extracted was insufficient for the amplification process. The damage of DNA molecules before being amplified due to some contaminants and the occurrence of porphyrin molecules in samples might also decrease the sensitivity and specifity of PCR, as stated Long (1990). According to Grimwood and Smith (1992), porphyrin is biomolecule sensitive to light and has electron power that may interfere electrophoresis process. However, the result of this research is a bit different from what had been done by McLeod et al. (1991), in which the difference is found in the infection method and the stages used to infect the Balb/c mice and also the timing when the DNA is detected.
Positive results obtained by PCR examinations on Day-7 and Day-9 after infection from all samples (liver, lung, heart, kidney and brain) were in agreements with positive results of tachyzoits isolation from peritoneal samples at the same days showing parasitemia ( Figure 2 and Table 2).
According to Cheng (1986), the selection of tissue samples (brain, liver, lung) for PCR examinations is suitable for evaluating the tissue on T. gondii infection. However, in this research, the result from examination of heart sample was also positive. It was probably due to all samples had already been infected since the mice treated with oocysts which had been sporulated in drinking water. Table 2 shows results of examination on liver, lung, heart, and kidney using histological approach, showing that cysts was only found in brain tissue (Figure 3). Microscopic examinations on other organs, however, only show infiltration of mononuclear cells (Figure 4). This was contrast to results of PCR examinations where all organs showed positive results. It can be concluded that PCR method has higher sensitivity than histological examination.
The infiltration of mononuclear cells visible in all tissues probably happened when infection was still active in an acute stage. Meanwhile, at chronic stage (bradyzoite stage), cysts inside the tissue was inactive and non-infective, and thus tissue reaction no longer occurred, and the parasite appeared in the form of cysts. Parasites did not show any reaction at chronic stage; therefore, although they stayed inside our body, they were relatively invisible because they were too small or because they lacked attention during the cysts formation (Remington and Desmont, 1990). This is very obvious in the figure showing no inflammation in the cysts of the tissue found in each organ.
From Figure 1 above, T. gondii tachyzoite in mice's ascites can be seen. It explains that tachyzoite in ascites according to Frenkel (1990) at an acute infection grows rapidly. In host cell, tachyzoite is accumulated in parasitophorus vacuole. Parasites can grow at all types of eukaryotic cells and multiply in a non-eukaryotic cells. However, in a medium without cell, tachyzoite cannot multiply and die after 6 hours. Multiplication of tachyzoite occurs by endodyogeny, and it results in the accumulation of 8 or more parasites gathered in one host cell. When the host cell breaks, the tachyzoite will be out and infect the new cell (Long, 1990). According to (Grimwood and Smith, 1992), the tachyzoite stage is known as invasive stage. When the conoid emerges, tachyzoite rapidly invades the cell and enters parasitophorus vacuole. Anterior organ, micronema and rhoptries are naturally seen as sectrory organelle which produces substance to help penetrate into the host cell. It is proven by the state in which zoites are within the host cells. Parasitophorus vacuole is a safe place for the growth of the parasite since it does not create fusion with lysosom and acidification which can destroy the parasites.

Conclusion
From the results and discussion, this research it can be concluded that: 1. PCR examination developed successfully amplified 436 bp DNA of T. gondii from some organs such as liver, lung, heart and brain on Day-7 and Day-9 post infection. 2. Using histological examination, cysts was only found in the brain while the other organs only showed mononuclear cells infiltration.


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Potential of Camellia sinensis proanthocyanidins-rich fraction for controlling malaria mosquito populations through disruption of larval development

Background Anopheles arabiensis and A. gambiae (sensu stricto) are the most prolific Afrotropical malaria vectors. Population control efforts of these two vectors have been hampered by extremely diverse larval breeding sites and widespread resistance to currently available insecticides. Control of mosquito larval stages using bioactive compounds of plant origin has the potential to suppress vector populations leading to concomitant reduction in disease transmission rates. In this study, we evaluated the efficacy of Camellia sinensis crude leaf extract and its fraction against the larvae of A. arabiensis and A. gambiae (s.s.). Methods Late third/early fourth instar larvae (L3/L4) of A. arabiensis and A. gambiae (s.s.) were exposed to increasing doses of C. sinensis leaf extract and its active fraction for 72 h, with mortality rates recorded every 24 h in both control and test groups. Ultra performance liquid chromatography electron spray ionization quadruple time of flight coupled with mass spectrometry (UPLC/ESI-Qtof/MS) was used to determine the main active constituents in the fraction. Results The major bioactive chemical constituents in the C. sinensis leaf extract were identified to be proanthocyanidins. The extract significantly interfered with larval survival and adult emergence in both species (ANOVA, F(5,24) = 1435.92, P < 0.001). Additionally, larval exposure to crude extract at 250 ppm and 500 ppm for 24 h resulted in larval mortality rates of over 90 % in A. gambiae (s.s.) and 75 % in A. arabiensis. A relatively lower concentration of 100 ppm resulted in moderate mortality rates of < 50 % in both species, but induced growth disruption effects evident as abnormal larval-pupal intermediates and disrupted adult emergence. The estimated LC50 concentrations of the crude leaf extract against A. arabiensis and A. gambiae (s.s.) larvae at 24 h were 154.58 ppm (95 % CI: 152.37–158.22) and 117.15 ppm (95 % CI: 112.86–127.04), respectively. The bioactive polar fraction caused 100 % larval mortality in both vector species at 25 ppm. Conclusions Our findings demonstrate the potential of green tea extract and its active constituents in disrupting mosquito larval development. This could contribute to the control of mosquito populations and improved management of malaria.

Background
Anopheles arabiensis and A. gambiae (s.s.) are the principal malaria vectors in Africa accounting for 88 % of the newly-reported global malaria cases and 90 % of the estimated death toll in 2015, impeding socio-economic growth in resource-constrained developing countries [1]. The diversity of mosquito breeding habitats hinders larval control efforts [2]. Currently, malaria vector control relies on integrated vector management (IVM) approaches such as targeting adult stages using long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS), environmental management and larval source reduction [1,3,4]. Substantial reduction of mosquito populations has been achieved by targeting behavioral attributes of the adult mosquitoes and destruction of larval breeding sites [5]. However, evolution of insecticide resistance in mosquito vectors due to persistent application of chemical insecticides poses a great threat to elimination of malaria from endemic regions [6,7]. Virtually all the vector control strategies have limitations prompting the need for application of multiple control methods to reduce malaria transmission rates as well as offset insecticide resistance [8,9]. Thus, there is renewed interest in the search for novel chemicals that have reduced chances of development of resistance, especially ecofriendly natural compounds that are less toxic, or selectively toxic to mosquitoes [10].
The immature mosquito stages breed in diverse habitats that may influence their vector competence [20,21]. Biophysicochemical parameters of breeding habitats have effects on the larval productivity and vector population dynamics [22]. Perturbation of these habitat parameters with bioactive agents has been reported to significantly suppress mosquito populations [23] and can negatively impact vector competence and life history traits of resultant adult mosquitoes [24,25]. Thus, targeting the immature stages of mosquitoes can be a viable approach towards elimination of malaria [26,27].
Several studies have focused on the pharmacological potential of green tea phytochemicals particularly prevention and treatment of cancer, microbial infections, malaria, arthritis, cardiovascular diseases, inflammation, neurodegenerative diseases and other oxidative stress related diseases [28]. Currently, little is known about their potential role in control of medically important insect vectors. Our present study was motivated by previous reports that demonstrated the inhibitory effect of green tea extracts on larval development in Drosophila melanogaster, Aedes aegypti, Ae. albopictus and Culex quinquefasciatus [29,30]. Further, green tea polyphenols have been shown to cause deleterious effects on the development and reproduction fitness of D. melanogaster [31]. Therefore, we designed a study to evaluate the efficacy of green tea leaf extract and its constituents against immature larval stages of A. arabiensis and A. gambiae (s.s.). Larvicidal and growth disruption effects of crude green tea leaf extract and its bioactive fraction against the mosquito immature stages were assessed. Our findings show that proanthocyanidins present in green tea extract reduced larval survival and adult emergence in both mosquito species by 100 % at 25 ppm. A sublethal dose of 5 ppm induced growth disruption effects resulting in abnormal larval-pupal intermediates and abortive adult emergence.

Experimental insects
Late third and/or newly emerged fourth instar (L3/L4) larvae of A. arabiensis and A. gambiae (s.s.) were used for experimental studies. These larvae were obtained from a mosquito culture maintained at the International Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya. To establish an experimental larval colony, eggs oviposited on moist filter papers were placed into hatching trays containing 0.5 L dechlorinated water at 28 ± 2°C. Following egg hatching, the mosquito larvae were kept in plastic trays (39 W × 28 L × 14 D cm) at densities of approximately 300 larvae per litre of distilled water and maintained under the following environmental conditions; 28 ± 2°C water temperature, 12 L:12D photoperiod and 55-60 % relative humidity. Larvae were fed with 0.3 mg Tetramin® fish meal (Tetra GmbH, Melle, Germany) once every 3 days.

Collection of plant material and extraction
Fresh immature leaves of green tea, C. sinensis (clone TRFK 6/8), were collected from Limuru Archdiocesan Farm (Limuru, Kenya; GPS coordinates: 01°07′10″S, 036°3 9′37″E; 2,225 m above sea level) in February 2016 with permission to use the plant for this study from Archdiocesan farmers. The tea leaves were shade-dried at 25 ± 2°C with intermittent aeration to a constant weight. Air-dried tea leaves were milled into powder using an electric grinder (Model 5657; Retsch GmbH, Haan, Germany). Five hundred grams (500 g) of the pulverized leaf powder was infused in 2 L of 90 % methanol for 72 h with intermittent shaking. The extract was filtered with Whatman 1 filter paper (Whatman Inc., Haverhill, USA) and excess solvent removed via rotor evaporation. The residual extract was lyophilized in a freeze dryer (Labconco stoppering tray dryer, Labconco Corporation, USA) programmed at 13°C temperature, vacuum pressure of 998 × 10 -3 millibars and collector at -40°C. The resultant extract was stored at -20°C until use. Based on TLC monitoring and evaluations, fractions with similar retention factor (Rf ) values were pooled together, rotor-evaporated and assayed for activity against mosquito larvae.

Larvicidal activity of green tea extract
Larvicidal assays were conducted following the World Health Organization (WHO) guidelines [32]. Batches of twenty (20) L3/L4 instar mosquitoes were placed in 250 ml glass beakers containing 100 ml of different concentrations of leaf extract (i.e. 500 ppm, 250 ppm, 100 ppm, 50 ppm and 25 ppm) and its fraction (25 ppm, 10 ppm, 5 ppm, 2.5 ppm and 1 ppm). Five replicates were set for each treatment dose (n = 100 larvae) and an untreated control (n = 100 larvae) was included in each experiment. The doses were formulated by separately dissolving 250 mg, 125 mg, 50 mg, 25 mg and 12.5 mg of leaf extract and 12.5 mg, 5 mg, 2.5 mg, 1.25 mg and 0.5 mg of active fraction in 1 ml of reagent grade absolute ethanol (Fisher Scientific, Loughborough, UK) and diluting into respective doses with 499 ml distilled water. The negative control experiment was set up by placing larvae in 0.2 % of ethanol diluted in distilled water.

Quantification of larval mortality rates
Equal starting numbers of larvae (n = 20 larvae) were placed into each beaker containing different concentrations of leaf extract and active fractions. Mortality rates of treated larvae were quantified at 24 h intervals. Each larva was examined and considered dead if it did not respond to probing with a dropper. Morphological defects of larvae induced by treatment with tea leaf extract relative to controls were analyzed using light microscopy at 25× magnification (Leica Corporation, Heerbrugg, Switzerland). High resolution images of larvae were captured and recorded for further analysis.

UPLC/ESI-Qtof/MS analysis
In order to re-dissolve the bioactive fraction, 1.5 mg was mixed with 1 ml of LC-MS grade CHROMASOLV methanol (Sigma-Aldrich, St. Louis, USA) and centrifuged at 14000× rpm for 5 min, after which 0.2 μl of the supernatant was automatically injected into UPLC/ESI-Qtof/ MS. The chromatographic separation was achieved on a Waters ACQUITY UPLC (ultra-performance liquid chromatography) I-class system (Waters Corporation, Milford, USA) fitted with a 2.1 mm × 50 mm, 1.7 μm particle size Waters ACQUITY UPLC BEH C18 column (Waters Corporation, Dublin, Ireland) heated to 40°C and autosampler tray cooled to 5°C. Mobile phases of water (A) and acetonitrile (B) each containing 0.01 % formic acid were employed. The following gradient was used: 0-5 min, Potential assignments were calculated using mono-isotopic masses with a tolerance of 10 ppm deviation and both odd-and even-electron states possible. The number and types of expected atoms was set as follows: carbon ≤ 100; hydrogen ≤ 100; oxygen ≤ 50; nitrogen ≤ 6; sulfur ≤ 6. The UPLC/ESI-Qtof/MS data acquisition and analysis were based on the following parameters: mass accuracy (ppm) = 1,000,000 × (calculated mass-accurate mass)/calculated mass; fit conf % is the confidence with which the measured mass (accurate mass) matches the theoretical isotope models of the elemental composition in the list; elemental composition is a suggested formula for the specified mass. This reflects a summation of the quantities of elements, isotopes or superatoms that can comprise the measured data, calculated using the following atomic masses of the most abundant isotope of the elements: C = 12.0000000, H = 1.0078250, N = 14.0030740, O = 15.9949146, S = 31.9720718. The empirical formulae generated were used to predict structures and proposed based on the online databases (Chemspider, Metlin) and published literature [33,34].

Data analysis
Data were entered and organized in Microsoft Excel 2010 spreadsheet then exported to R software version 3.2.3 (The R Project for Statistical Computing, www.r-project.org) for analyses. Corrected mortality rates were expressed as % mean ± standard deviation (SD) for each tested dose. The test concentrations were log 10 -transformed to reduce variation prior to fitting a dose-response model for estimating lethal dose concentrations. Nonlinear regression using glm function in R with probit link and quasi binomial distribution error was used to estimate the lethal concentrations of crude extract and its active fraction. LC 50 of both the crude extract and its active fraction were estimated from the glm output using the dose.p function in MASS Package in R. The significance of differences between treatment means was determined by analysis of variance (ANOVA) with values of P < 0.05 considered statistically significant. Graphs were designed using GraphPad Prism version 7.0 for Windows (GraphPad Software, San Diego, USA). Images were processed using publicly-available IMAGEJ software (National Institutes of Health, https://imagej.nih.gov/ij/).

Toxicity effects of leaf extract and its fraction on mosquito larvae
Larval survival and adult emergence was significantly reduced when L3/L4 instars of A. arabiensis and A. gambiae (s.s.) were exposed to different concentrations of crude extract and its bioactive fraction (ANOVA, F (5,24) = 1435.92, P < 0.001). The untreated control group achieved 100 % survival rates for the entire analysis period. Higher extract Table 1 Tentative identification of the constituents of C. sinensis bioactive fraction. Data show tentative identification of compounds within the bioactive fraction of C. sinensis extract from published literature and publicly accessible online databases, monoisotopic mass m/z, chemical formula, and peak area of each compound at a particular retention time (R t )  Table 2). A sublethal dose of extract at 100 ppm induced growth disruption effects characterized by: failure of larvae to molt into pupae, resulting into abnormal larval-pupal intermediates (Fig. 3c, d) and death of adults during eclosion from the pupal stage, with their legs and wings stuck in pupal caste (Fig. 3e, f). Further, we investigated the larvicidal efficacy of the bioactive fraction by exposing the larvae to 25 ppm, 10 ppm, 5 ppm, 2.5 ppm and 1 ppm. Maximum larval mortality rates of 100 % were attained at 25 ppm treatment dose within 24 h incubation in both A. arabiensis and A. gambiae (s.s.) (Table 3 and Fig. 2). Moderate larval toxicity that increased with incubation duration reaching peak levels at 72 h post-exposure occurred at 2.5, 5 and 10 ppm, whereas minimal toxicity was recorded at 1 ppm exposure. Growth disruption effects similar to those induced by the crude extract at 100 ppm were observed at 5 ppm (Fig. 3). Differences between the treatment means were not statistically significant (ANOVA, F (5, 24) = 85.33, P < 0.001).
Treatment of mosquito larvae with sublethal doses of green tea bioactive fraction exerted growth disruption effects after 72 h of exposure (Fig. 3). Progression of metamorphosis, from larva to pupa and finally adult, was halted resulting into abnormal larval-pupal intermediates and arrested adult emergence. Further, some adults that successfully emerged from the larvicide treated water were unable to fly from the test beakers and died on the water surface, a phenomenon similarly observed with crude extract at 100 ppm.

Discussion
Malaria-transmitting mosquitoes have developed physiological and behavioral mechanisms to successfully evade population control strategies targeting adult stages [7]. Limitations imposed by these vector control tools have necessitated complementary strategies. Larviciding, a lesspracticed component of integrated vector management, has traceable history of success in eradication of malaria from Brazil and Egypt and currently revived in some African countries [26,35]. Mosquito metamorphosis represents a low cost and feasible target of population regulation through interfering with development of immature stages [36]. Despite the dramatic reduction in mosquito populations on persistent application of chemical insecticides, negative implications have limited their continued use [37]. Thus, development of safe, novel and selectively toxic larvicides has been welcomed into integrated mosquito control programmes [38,39]. Principally, their anticipated multimodal actions against target arthropods limit chances of resistance development and could be a pathway to designing resistant-resilient insecticides [40]. Prospection for use of plant-derived insect growth regulators as biolarvicides has gained considerable attention aimed at disrupting successful development of agricultural and medical nuisance insects including mosquitoes [10,13,41].
In this study, we aimed to evaluate whether green tea could be a potential source of mosquito control agents, by investigating its efficacy against larvae of A. gambiae (s.s.) and A. arabiensis. We found that, the initial larval bioassays with crude leaf extract significantly reduced survival and development of larval stages in both mosquito species Table 3 Acute toxicity in L3/L4 instars of A. arabiensis and A. gambiae (s.s.) resulting from treatment of larvae with the active green tea fraction for 24, 48 and 72 h. Data presented below indicates the percentage means (± S.D) of mortality rates of mosquito larvae exposed to different concentrations of bioactive fraction of C. sinensis extract for different time periods (24, 48 and   suggesting presence of biologically active phytochemicals. This necessitated fractionation in order to isolate specific bioactive ingredients, tentatively identified as caffeine, quercetin, kaempferol, kaempferol rhamnoside, kaempferol rhamnosyl glucoside and proanthocyanidins. Consistent with previous results from studies conducted on other insects [29][30][31], C. sinensis extract exhibited larvicidal activity in addition to induction of growth disruption effects. However, in contrast to our dosage data, these studies reported relatively higher extract dosages of between 10 mg/ml to 75 mg/ml (translating to 10,000-75,000 ppm) to achieve similar larvicidal potency. The varied observations could be attributed to susceptibility difference of test insects, variation in abundance of bioactive constituents in plant extracts, extraction method and geographical location of the studied plants. Contrary to these studies which attributed toxicity effects to polyphenolic constituents of C. sinensis especially the abundant (-)-epigallocatechin-3gallate (EGCG) [31], we found proanthocyanidins to be the abundant bioactive compounds most strongly associated with the observed effects. Surprisingly, catechins were not detected in the bioactive fraction. However, proanthocyanidins are polyphenolic products of catechins epimerization associated with plant defenses [16,19]. The other compounds identified within the fraction might have contributed towards exerting synergistic or racemic mixture effects to proanthocyanidins.
Caffeine has been reported to interfere with mosquito larval development [42]. The hydroxylflavones, quercetin and kaempferol, induce cell cycle arrest by inhibiting CDC25A tyrosine phosphate at G2/M phase and/or inducing apoptosis [43]. In nature, polyphenolic compounds including proanthocyanidins (condensed tannins) and other flavonoids form part of defense against fungal  attacks and insect herbivory in plants [44]. They provoke feeding deterrence with intense disorganization of midgut epithelia cells upon ingestion, which concomitantly reduces insect survival and development [45]. The fact that green tea polyphenolic compounds exert anti-carcinogenic effect by inducing cell cycle arrest, apoptosis and growth inhibition could also be implicated in this study [46,47]. The post-embryonic stages of insects comprise of cell proliferation and DNA replication events preceding growth and morphogenetic organization [48]. As proanthocyanidins are pro-oxidants and pro-apoptotic molecules that astringently precipitate cellular proteins [49], they could presumably halt these events in developing insects resulting to death. Also, the compounds bind to nucleic acids, increasing topoisomerase II DNA cleavage activity, inducing DNA breaks and reduced cell survival [50,51]. Taken together, these mechanisms could be attributable to the impaired larval development and toxicity.
Although molecular studies were not included in this study to determine the linkage between the genotype and the observed morphological traits, peculiar observations were noted in regard to developmental phenotypes. Incubation of the mosquito larvae with sublethal dose of 5 ppm of the bioactive fraction induced developmental defects (Fig. 3) similar to those exhibited by insect growth regulators (IGRs) [52,53]. The transition of mosquito larvae into adult stage was found to fail at larval-pupal intermediates, while others remained permanently stuck as they eclosed. Insect growth regulatory (IGR) compounds act by adversely interfering with physiologically and hormonally-regulated developmental events resulting into immature deaths and non-viable reproductive adults [52,54]. Commonly known IGRs are insect developmental hormone agonists that mimic the actions of juvenile hormone and ecdysone while others inhibit chitin synthesis [55,56]. Interestingly, among the compounds tentatively identified in the bioactive fraction, none seemed structurally similar to insect developmental hormones, ecdysteroid 20-hydroxyecdysone (20-E) and sesquiterpenoid juvenile hormone (JH). However, the presence of flavonoid-like polyphenols in the larval breeding water suggested modulated neuroendrocrine signaling networks interfering with larval development [57]. Of importance is insulin/insulin-like pathway, a conserved regulatory signaling pathway that coordinate insect growth and metamorphosis by regulating biosynthesis of development hormones [58][59][60]. Other studies have documented that phytochemicals especially flavonoid-like polyphenols negatively impact insect molting by interfering with prothoracicotropic hormone and ecdysteroid action resulting to delayed maturity and abnormal developmental phenotypes or mortality [61]. Growth and development transitions in immature insects are orchestrated by morphological and ultra-structural changes regulated by coordinated actions of JH, ecdysone and eclosion hormones [62]. Insulin/insulin-like signaling interplay between the developmental events to ensure static allometry in holometabolous insects [63][64][65]. Hence, any exogenous agent that interferes with either the signaling networks or homeostasis of the insect developmental hormones might result in abnormal growth and development as observed in Fig. 3.
Plant-based IGRs with similar effects on developing mosquitoes have been previously reported to effectively suppress mosquito populations [66][67][68][69][70][71]. C. sinensis is favoured over other plants as a potential source of mosquito control agents because it is (i) cultivatable (ii) non-endangered plant species, and (iii) its waste can be recycled. Our findings suggest that green tea leaf proanthocyanidins could be employed to control malaria vector populations. It is paramount to conduct studies to determine toxicity effects of proanthocyanidins against non-target organisms prior to commercialization and large scale field application of this product as larvicidal agent. Further, studies encompassing molecular target identification, simulation and ligand docking are recommended to create avenues for synthesizing structurally similar candidate analogs to ease application.

Conclusion
In Kenya, C. sinensis is grown as a major cash crop. We found that the leaf extract and its active constituents have great potential for controlling mosquito larvae. Proanthocyanidins were the abundant compounds tightly associated with toxicity of larvae though the other compounds identified in the extract may contribute to bioactivity. Besides causing larval toxicity, the sublethal doses induced growth disruption effects that inhibited adult emergence.