Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and/or with novel drugs targeting other survival pathways in MM. The rationale for targeting the Pims in MM, lead Pim inhibitors in development and the potential application of Pim inhibition in treatment of MM are discussed herein.The Pim kinases have emerged as an attractive novel therapeutic strategy in the hematologic malignancies in general, and in multiple myeloma (MM) in particular. Pims are constitutively expressed uniquely in the hematologic malignancies and Pim-2 expression is higher in MM than in any other cancer or in physiology.8 Insertional mutagenesis screening utilizes transforming retroviruses to identify oncogenes overexpressed by the activity of the retroviral enhancer sequence.9 Cloning of retroviral integration sites in mMuLV-induced lymphomas led to the discovery of Pim-1 in the 1980s8 followed by Pim-210 and later Pim-3 in the 1990s in the screening of Pim-1/Pim-2 knockout models.11The Pim family of serine/threonine kinases are named for their mode of discovery as proviral common integration sites in moloney murine leukemia virus (mMuLV)-induced lymphomas.12 and the genetic structure is outlined in 14 Pims have a unique structure divergent from that of other kinases with two proline residues located in the hinge region.13 Only one hydrogen bond is formed with ATP, with implications for drug development as the majority of ATP-competitive inhibitors form two. The Km  of Pim-2 for ATP is up to 100-fold lower than that of Pim-1 and Pim-315 making a pan-inhibitor more challenging to develop than specific inhibitors.16The Pim family is highly conserved with greater than 60% homology between each member17 and nuclear factor Kappa-light-chain-enhancer of activated B cells (NFκB)18 pathways results in transcription of Pim mRNA. STAT3 and STAT5 levels correlate with Pim expression.14Regulation of Pim kinases is predominantly transcriptional and translational. Growth factor signaling via the janus kinase/signal transducer and activator of transcription (JAK-STAT)19 The Pim 5' region is GC sequence-rich and hence is a ‘weak transcript' requiring cap-dependent translation (see section 'Pim kinases and cancer—Cap-dependent translation').20 Pims are capable of autophosphorylation at serine8,21 but the stability of the transcribed PIM proteins is the key regulator of Pim activity. Pims are dephosphorylated, ubiquitylated and directed for proteasomal degradation by the B56β protein phosphatase 2A (PP2A).22 Members of the heatshock protein family have opposing roles in the regulation of Pim activity with HSP90 stabilizing Pim levels and HSP70 marking Pims for ubiquitylation and proteasomal degradation.23Pim mRNA has a short half-life because of the presence of the destabilizing AUUU(A) sequence at the 3' region, though in hematologic cancers longer half-lives are observed.Pim1−/− Pim2−/− Pim3−/−) resulted in mild phenotypic changes in murine studies. Mice were viable, fertile but exhibited reduced body size.24Initial studies involving knockout of all three Pim kinases  and chronic myeloid leukemia.1 Pim-3, while expressed throughout the hematologic malignancies, shows no distinct pattern of expression favoring a particular malignancy1 and bears the least association with hematological malignancies, instead being strongly associated with solid organ malignancies.28 Expression of Pim kinases confers a worse prognosis in many malignancies, including AML.12Pim kinases are widely expressed in cancer with higher expression in hematologic than solid organ malignancies.6 and in turn, the Pims phosphorylate, stabilize and enhance the transcriptional activity of Myc, an effect greatly diminished by Pim knockdown.29 Pim kinases complex with Myc and Myc-associated factor X(MAX), with Myc recruiting Pim to the E-box of target genes.30 Pim kinases then phosphorylate serine10 of histone H3 (H3S10) of the Myc binding site nucleasome, increasing transcriptional activation of approximately 20% of Myc-targeted genes and contributing to cellular transformation.30 H3S10 phosphorylation facilitates interaction with 14-3-3 proteins which trigger the acetylation of histone H4, and this crosstalk between the histones provides a platform for bromodomain protein binding.31 The ensuing recruitment of transcription elongation factor b (TEFb) releases promoter-proximal paused RNA polymerase II and activates transcription.31Myc activity is necessary for Pims to effect oncogenesis,32 Pim-1 transgenic mice with Eμ Immunoglobulin enhancer sequences upstream of the Pim gene and long terminal repeats downstream exhibited greatly increased expression of Pim-1 with a corresponding increase in lymphomagenesis.33 Newborn Pim-1 transgenic mice infected with mMuLV with activation of MYC developed T-cell lymphomas with a latency of only 7–8 weeks,33 evidencing a very strong oncogenic collaboration between these genes. While Pim-1 and MYC transgenic mice were predisposed to T- and B cell lymphomas, respectively, double transgenic Eμ Pim-1 and Eμ MYC developed an aggressive pre-B leukemia in utero with death at or before birth.34The originally described mMuLV-induced T-cell lymphomas exhibited highly increased c-MYC expression, with proviral insertions frequently occurring in close proximity to both c-MYC and Pim-1 genes.11 In support of redundancy of Pims in hematological malignancy, upregulation of Pim-2 in Pim-1-deficient mice and Pim-3 in Pim-1/Pim-2-deficient mice with preserved lymphomagenesis is observed.11Further evidence for this Pim/MYC collaboration and MYC dependence on Pim expression for oncogenesis is provided by observation of longer latency to development of lymphoma in Pim knockout mice.1 mTORC1 then phosphorylates EIF4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase (S6K).Pims are important in the upregulation of proteins involved in cell cycle regulation and cell survival via cap-dependent translation in cancer. Pim-2 phosphorylates tuberous sclerosis complex-2 (TSC2) to derepress mammalian target of rapamycin complex-1 (mTORC1).7G-capped mRNA for translation. EIF4E is necessary for Pim-induced cap-dependent translation to occur.35 Furthermore, the activation of EIF4E following mTORC signaling is crucial for MYC survival signaling.36 The Pims themselves, as well as MYC, cyclin D1, myeloid cell leukemia-1 (MCL-1), important in survival and cell cycle progression, constitute ‘weak' mRNA targets owing to their 5' GC-rich region37 and rely on this mechanism of translation.Phosphorylation of 4EBP1 facilitates separation from EIF4E and allows recruitment to ribosomal subunit 40S of m38 In chronic lymphocytic leukemia, Pim inhibition at concentrations sufficient to reduce MYC and MCL1 expression affects cell death, whereas antiapoptotic effects were not affected at this level. Similar data are presented relating to MM, indicating a dominant role for blockade of Pim-2-induced cap-dependent translation in clinical use of Pim inhibitors in lymphoid malignancy.39In B-cell lymphoproliferative malignancies, Pim-2 has a dominant role by regulating mTORC1, as evidenced by reduced phospho-4EBP1 with Pim inhibition.14 Multiple sites on BAD may be phosphorylated to prevent apoptosis,40 with S112 phosphorylation, the dominant residue involved in Pim-1 and Pim-2 signaling, and Pim-3 favoring S136 and S155 phosphorylation.40 Following phosphorylation, 14-3-3 binding occurs with dissociation of BAD from B-cell Lymphoma-extra large prosurvival protein (Bcl-XL) and relocation from the mitochondrion to the cytosol.40 Other anti-apoptotic activities include phosphorylation of murine double minute 2 homolog (MDM2) at serine166 and 186 to prevent proteasomal degradation of p53 in mantle cell lymphoma41 and inhibit the apoptotic actions of apoptosis signal-regulating kinase-1 (ASK-1) by its phosphorylation.42The best described anti-apoptotic effect of the Pims is that on Bcl-2-associated agonist of cell death (BAD) phosphorylation. This effect was initially discovered in Pim-2.44 Phosphorylated p21 relocates to the cytoplasm and is stabilized to increase proliferation.43 By contrast, Pim phosphorylation of p27 induces 14-3-3 binding, its transport from the nucleus and proteasomal degradation.44 Pims also inactivate Forkhead transcription factors to downregulate p27.44 Pims target the G1/S transition point by phosphorylating CDC25A, a transcriptional target of Myc and increasing cyclinD1 activity.4 Pims regulate the G2/M checkpoint by phosphorylating CDC25C and CDC25C-associated kinase 1.3 Pim-1 and -2 have also been found to phosphorylate Chk1 at serine280 in AML,45 perhaps as a component of the FLT3/STAT5/PIM/CHK1 axis, though increased Chk1 phosphorylation is also observed in Flt3 wild-type AML primary samples,45 and inhibition of Pims in wild-type or mutated Flt3 AML appears to result in similar inhibition of proliferation.The Pim kinases phosphorylate cyclin-dependent kinase inhibitors p21 and p27.5 and strong oncogenic collaboration between the Pims and Myc, Pim inhibitors seem an obvious choice for drug development in MM. In addition, Pims crosstalk with and share significantly overlapping functions with other kinase signaling pathways active in MM.Pims recently emerged as an exciting new target in MM. High expression of Pim-2 is seen in MM and important in mediating MM cell survival and proliferation, by inhibiting apoptosis and inducing cap-dependent translation, respectively . Further1 Microarray analyses identify Pim-2 as a key player in B-cell development as well as MM.46 High expression is not observed in normal plasma cells and expression increases in MM versus monoclonal gammopathy of unknown significance.47 This infers a role for Pim-2 in progression to MM, consistent with observations in other lymphoid malignancies of increasing Pim-2 expression with advancing disease.49 At the time of writing, there were no published data to establish a worse prognosis conferred by increased Pim expression in MM, though this has been seen in other lymphoid malignancies.38Pim-2 is more highly expressed in MM than in any other malignancy.49 The bone marrow microenvironment has a dominant role in upregulation of Pim-2 in MM. Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) elaborate specific growth factors to increase the expression of Pim-2 in MM cells.2 BMSCs have a more pronounced effect, with a two to ninefold increase in Pim-2 mRNA following BMSC coculture of MM cells mediated by production of interleukin (IL)-6, which acts via STAT3 to increase transcription of Pim-2.2 OCs increase Pim-2 expression via the tumor necrosis factor family members TNFα, BAFF and APRIL, which act by NFκB signaling to increase transcription.2 NFκB is necessary for Pim-2 anti-apoptotic effect, and Pim-2 in turn increases phospho-IκB and Cot transcription factor to increase NFκB, in a positive feedback loop.50 In support of these mechanisms of Pim-2 upregulation in MM, inhibition of STAT3, NFκB and IL-6 all reduce Pim-2 expression in in vitro models.2 In contrast to IL-6 and NFκB, IGF-1 has not been found to have a significant role in mediating Pim-2 expression via the microenvironment2 as it does with other signaling pathways in MM. Cooperative enhancement is observed with a combination of BMSCs and OCs in the microenvironment increasing Pim-2 expression significantly. Furthermore, siRNA knockdown of Pim-2 significantly abrogates MM cell viability in coculture with BMSCs and OCs,2 confirming the anti-apoptotic role of Pim-2 in MM.Pims are constitutively active following growth factor signaling in MM, with no mutated forms reported to date, though a high rate of somatic hypermutation involving the Pim genes is reported in other B lymphoproliferative malignancies.1 Pim-2 phosphorylates the negative regulator of mTOR, TSC2, at serine 1798 to relieve its suppressive effect on mTORC1 activity in MM.1 Phosphorylation of S6K and 4EBP1, substrates of mTORC1 signaling, increases following Pim-2 signaling, facilitating cap-dependent translation and proliferation by the mechanism already described. There is evidence from preclinical work in MM using Pim inhibitors that inhibiting this process is the key anti-MM effect of these drugs.Pim-2 is necessary for proliferation in MM.1 Pim inhibition with thiazolidine class reduces 4EBP1 phosphorylation and the amount and phosphorylation of MCL1 and c-MYC in MM cell lines.2 Reduced anti-apoptotic phospho-BAD is seen following Pim-2 inhibition with this drug, or Pim-2 knockdown, but the effect is less pronouned than that of reduced proliferation.1 Pharmacological inhibition with SGI-1776 results in no change in apoptosis or cell cycle regulation,51 but protein tranlsation is affected, with reduced phosphorylation of 4EBP1 and P70S6K.51 Furthermore, in MM animal models, maximal in vivo effect is dependent on the inhibition of mTORC1 signaling, and hence inhibition of proliferation, being potent and sustained.1Pim inhibition with LGB321 reduces phosphorylated TSC2 and mTORC1 activity.7 Pim-2 is seen to negatively regulate osteoblastogenesis in murine cell line models, with the effect reversed on Pim-2 knockdown or pharmacological inhibition.7 The chief osteoblastogenic effects mediated by Pim inhibition were increase of osteoblastogenic factor bone morphogenetic protein-2 (BMP-2) and reduced transforming growth factor β signaling. Pim-2 expression is upregulated not only in MM cells but also in BMSCs and osteoblast precursors in the microenvironment in response to known suppressors of osteoblastogenesis IL-3, IL-7, Activin A, TNFα and transforming growth factor β, and this effect was also noted in primary human MM BMSCs following culture with conditioned media.7 Pim inhibitors are capable of counteracting these effects to restore osteoblastogenesis. Murine models of human MM treated with Pim inhibitor SMI16a reveal reduced tumor growth, prevention of bony destruction and restoration of bone formation compared to control animals.7 The Pim inhibitor LGH447 reduces bone disease in MM xenograft models 52 and reduces formation and functionality of OCs.53An emerging role for Pim-2 in the bony destruction and marrow expansion of MM has been reported.54 It has been shown that SDF1α and CXCR4 interactions mediate MM expansion and homing55 and that inhibition of CXCR4 inhibits MM homing.56 The MM bone marrow microenvironment is a hypoxic niche.57 Hypoxia reduces MM adhesion to bone marrow stroma by reducing E-cadherin expression following increasing expression of SNAIL, FOX2C and transforming growth factor 1β.58 Hypoxia also results in reduced SDF1α production by stromal cells locally,58 potentially encouraging migration and homing to new sites in the marrow. CXCR4 levels are higher in circulating MM cells than in those resident in the bone marrow.54 Further, hypoxia induces upregulation of CXCR4 in a HIF1α-dependent manner in MM.58 The hypoxic bone microenvironment conditions also promote Pim activity with inhibition of the ubiquitin-mediated proteasomal degradation of Pim.59MM is characterized by continuous migration of malignant cells from the bone marrow, and homing to new sites in the marrow.60 and confers a worse prognosis.61 Pim-1 phosphorylates Serine339 on CXCR4 facilitating receptor internalization and re-expression on the cell surface.60 Conversely, Pim inhibition results in reduced CXCR4 expression.60 In murine models, the knockout of Pim-1 results in the inability to reconstitute bone marrow following irradiation, and Pim-1 knockout cells express greatly reduced amounts of CXCR4.60 In chronic lymphocytic leukemia, a correlation is also seen between Pim-1 expression and CXCR4 surface expression.62 As in AML, Pim-1 phosphorylates Serine339 on CXCR4.62 Pim inhibition in chronic lymphocytic leukemia is capable of increasing CXCR4 internalization, reducing surface re-expression and migration to bone marrow and spleen.62 It is therefore plausible that Pim kinases may at least partially contribute to the upregulation of CXCR4 in the hypoxic microenvironment of MM and, by this mechanism, contribute to migration and homing of cells in MM also.It has been demonstrated in AML that Pim-1 is important for CXCR4 expression and homing of stem cells. A clear association between blast expression of Pim-1 and surface expression of CXCR4 has been established63 by altering phosphorylation status of relevant receptors, for example, androgen receptor in prostate cancer.64Pims have been described as having a role in anti-cancer drug resistance. This may be accomplished by increasing the expression of drug efflux pumps as in breast cancer,65 and combination with cytarabine in AML.66 Pim-2 confers resistance in hematopoietic cells to rapamycin inhibition of mTOR.67 Rationale for targeting the Pim and PI3K/AKT/mTOR pathways in combination is discussed below.In hematologic malignancies, targeting Pim kinases in combination with standard treatment has proved useful in overcoming resistance in preclinical models, for example, combination with JAK2 inhibition in MPN68 Knockdown of Pim-1 resensitizes cells to chemotherapy.68 Treatment with bortezomib increases Pim half-life by prevention of Pim proteasomal degradation,23 and it is possible that by this mechanism, Pims have a role in resistance to proteasome inhibitors in MM.It is highly likely that the hypoxic bone marrow microenvironment of MM has an important role in mediating drug resistance. Pim-1 has a role in hypoxia-induced chemoresistance in a HIF1α-independent manner by altering mitochondrial transmembrane potential and the activity of caspases-3 and -9.16The unusual structure of the Pim kinase ATP-binding site and challenges with inhibiting Pim-2 have been discussed. Given the redundant functions of the Pim family kinases established in lymphoid malignancies, it is conceivable that MM cells are capable of upregulating Pim-1 or Pim-3 as a survival mechanism if Pim-2 alone is targeted, and pan-Pim inhibition is most likely to be useful in the clinic. Structure- and property-based design approaches are actively used to identify inhibitors capable of targeting all Pim family members with potency in the nanomolar range.In vitro work in MM cell lines revealed cytotoxicity in Pim2-dependent MM by inhibiton of mTORC1 with reduced phosphorylation of 4EBP1 and S6K; promoting apoptosis with inhibition of BAD phosphorylation and cleavage of PARP and caspases 3, 7, 8 and 9; and cell cycle arrest at G0/G1.53In vitro work has also demonstrated an inhibitory effect of LGH447 on OCs, inhibiting their formation via reduced expression of phospho-ERK1/2 and NFATc1, and inhibiting resorptive function by reducing expression of matrix metalloproteinase, carbonic anhydrase II and the F actin ring necessary for functionality.53 Furthermore, reduced tumor growth and bone tumor burden was seen in human xenograft MM models following treatment with LGH447.52 LGH447 is active in the VK*MYC transgenic mouse model,69 which exhibits Myc dysregulation and is highly faithful to human MM, boasting positive and negative predictive values of 67% and 86%, respectively, for clinical effective of anti-MM treatments.70LGH447 is a potent pan-Pim inhibitor which has entered clinical phase development in MM See . In vitr71 Fifty-four patients received LGH447 as monotherapy with a median of four lines of prior treatment and 68.5% had been previously treated with both proteasome inhibitors and Imids. Response was evaluated in 48 with overall response rate  10.5%, clinical benefit rate (minor response or greater) 21.1% and an impressive disease control rate (stable disease or greater) of 71.9%. Maximum tolerated dose was 500 mg daily. Dose-limiting toxicities observed were grade 3/4 thrombocytopenia (4), grade 3 fatigue (2), grade 3 hypophosphatemia (1) and vasovagal syncope (1). Most adverse events were grade 1/2 and manageable, and no deaths occurred during the study.71 A phase I/II clincial trial of LGH447 in combination with the PI3K inhibitor BYL719 in relapsed/refractory MM is ongoing (NCT02144038).Results of a first-in-human Phase I study of LGH447 in relapsed/refractory MM (NCT01456689) have recently been reported.72 LGB321 was identified during a screen for pan-Pim inhibitors and selected for further development because of a relative potency for inhibiting Pim-2 with its lower Km for ATP.16 Mechanistic studies indicate its cytotoxic anti-MM effect is mediated by inhibition of mTORC1 signaling and BAD phosphorylation.72 LGB321 was effective in a Pim-2-dependent MM xenograft model.1 To date, LGB321 has not entered clinical development.This potent ATP-competitive pan-Pim kinase inhibitor has activity against a broad range of hematologic malignancy cell lines including Pim-2-dependent MM.in vitro and in vivo settings in combination with Akt inhibitor AZD5363 in MM and AML.73 In MM, AZD1897 alone reduced proliferation of MM cell lines but did not have a dramatic effect on cell death, whereas in combination with the Akt inhibitor synergystic cytotoxicity was observed, with >50% cell death. Convergence on inhibition of BAD and MCL1 are the mechanism of synergystic cytotoxicity, as well as enhanced inhibition of mTOR signaling.73 AZD1897 has not yet reached clinical phase development.This ATP-competitive pan-PIM kinase inhibitor has been studied in As well as potential for combination with current standard of care in an attempt to overcome resistance/refractoriness, there is strong rationale for investigating Pim inhibitors in combination with inhibitors of the PI3K/AKT/mTOR pathway and MAPK/ERK pathway inhibitors.1 Akt signaling via PRAS40 exerts the same effect on mTORC1.1 Following this, not only is cell proliferation enhanced, apoptosis is also indirectly prevented by upregulation of MYC and MCL-1. Both pathways phosphorylate BAD to prevent apoptosis also. Pim kinases are capable of mediating resistance to the targeting of the PI3K/AKT/mTOR pathway.74 In lymphoid malignancies, Pims allow cells to maintain mTORC signaling in the context of mTOR inhibition.67 In prostate cancer, resistance to AKT inhibition is mediated by Pim-1 increasing translation of receptor tyrosine kinases in a cap-independent manner.74 Combinations of PI3K inhibitors and Pim inhibitors in MM in vitro work have shown synergy,1 and a recent abstract indicates synergistic cytotoxicity with the combination of AKT inhibitor AZD5363 and Pim inhibitor AZD1798 with convergence on inhibition of BAD and MCL-1 signaling outlined as key mechanisms,75 as was previously demonstrated in AML.73 This provides clear rationale for pursuing this combination into clinical studies.This pathway is highly expressed in MM and shares many downstream targets with the Pim kinases which function in parallel, though independently. In MM, the salient overlapping function is mTORC1 regulation. Pim kinases phosphorylate TSC2 to activate mTORC1 and also phosphorylate 4EBP1 with activation of EIF4E resulting in cap-dependent translation.76 Targeting this pathway proved effective in reduction of tumor burden in murine models.77 MAPK family members including ERK and JNK are upregulated in response to Pim signaling in prostate cancer,12 AML,78 hematopoietic cells79 and cardiomyocytes.80 The combination of Pim inhibition and MAPK inhibiton affected synergystic cell killing in T-cell leukemia.81 The risk of cardiotoxicity may be significant with this combination given the known risk of QTc prolongation with Pim inhibitors and the importance of MAPK signaling related to Pim expression in cardiomyocytes.This pathway is constitutively active in MM and implicated in MM-related bone disease.Much progress has been made in the treatment of MM to date, however, novel treatment approaches are urgently required to improve patient survival. It is hoped that further strides toward cure will be realized with emerging treatments such as Pim inhibition.12 suggesting that disease control, rather than significant tumor killing, is the realistic effect of Pims as single agents. The greatest effect of Pim inhibition in MM is on proliferation, which is unlikely alone to result in total elimination of the malignant clone. It is therefore expected that combination approaches will be necessary for Pim inhibition to provide significant survival benefit.Knowledge of MM, and of the biology of cancer in general, dictate that in response to small molecule inhibition of kinase signaling pathways, compensatory signaling can affect resistance. The Pim kinases are likely to prove no exception. Murine models of AML have demonstrated that a high tumor burden persists in spite of improved survival with Pim inhibition with rapid relapse on drug withdrawal23 lending rationale to combination of Pim inhibitors with proteasome inhibitors, a key standard component of MM treatment regimens.Combination of Pim inhibitors with current standard therapies capable of affecting cell death may prove an effective strategy. It is known that proteasome inhibition prevents degradation of Pim kinases,71 Other survival signaling pathways are simultaneously active in advanced disease and combining Pim inhibition with inhibitors of the PI3K/Akt/mTOR pathway in this setting constitutes another application for Pim inhibitors in MM. Where TORC1/TORC2 inhibitors have failed to translate to extended survival in the clinic, Pim kinases, particularly as combination treatments, may have more success. The Pim kinases themselves mediate resistance to mTOR inhibition in hematologic malignancies and may partly account for the lack of efficacy encountered.67 The converse may also be true, and it may prove necessary to inhibit the PI3K/Akt/mTOR pathway in tandem for durable results. Pim inhibition may have an added advantage over mTOR inhibition given the myriad other MM processes Pims are involved in.Pim expression in MM correlates with advanced disease, and clinical studies of LGH447 in relapsed/refractory MM are ongoing with data reporting impressive single agent activity.71 Avoidance of overlapping toxicities is a priority in the selection of suitable Pim inhibitor drug partners, with the propensity of Pim inhibitors to induce cardiotoxicity a key consideration. As inhibiting proliferation is the key to the anti-MM activity of Pim kinases, and can be achieved at doses lower than those required for induction of apoptosis, for example, utilizing the minimum dose required to achieve this anti-proliferative effect may limit potential for cardiotoxicity. The Pim inhibitors are also capable of suppressing hematopoiesis and selection of combination partners from currently available treatments should focus on less myelosuppressive agents.In general, Pim inhibitors demonstrate good tolerability and manageable side effects in the data reported.Ongoing clinical studies of LGH447 as a single agent and in combination with PI3K inhibitor BYL719 in advanced MM are anticipated to expedite the progression of Pim inhibitors with rational partners to the clinic and are eagerly awaited.


The role played by inorganic chemical additives in fire retardancy and flame inhibition is considered. Particular attention is given to the molecular level aspects of commercially important systems containing compounds of antimony, halogens, and phosphorus. The flame inhibiting function of metal containing additives is also discussed. It has long been recognized that the addition of chemical substances to combustible materials can considerably reduce the degree of flammability. In the specific case of textile fabrics, an extensive technology has developed through which a wide range of chemically distinct incorporants can be utilized to retard flammability while other desirable properties such as permanence of treatment, appearance, wear resistance, and hand texture are retained –61.“Although much can be deduced from the results of applied studies in the literature, there have been relatively few experiments conducted specifically to determine the mechanisms of retardance. At this stage, most of what’ can be said is highly speculative.”A well-developed empiricism has been elaborated which relates retardant characteristics such as elemental composition, chemical structure, and degree of loading to the reduction of flammability. The literature of this and related fields offers considerable deductive insight into the mechanisms by which fire retardants function. However, it is apparent, as Lyons  has statWhile the undoubted successes of applied chemistry in flame retardancy are clearly apparent, it has become increasingly important, as requirements for reducing the flammability of combustible materials become more stringent, to develop a detailed understanding at the molecular level of the mechanisms by which flame retardants operate. Knowledge of these essential chemical processes can, for example, permit more sophisticated design of retardants for particular applications and perhaps define the performance limits to be expected of particular retardant systems under service or test conditions. Candidate retardant materials can be selected more efficiently for screening if their chemistry can be related to the fundamental basis of retardant action. Retardants can be formulated that optimize performance and minimize undesirable side effects such as formation of smoke and toxic reaction products.The combustion of a fabric, or organic polymer, involves a preflame thermal degradation of the material, resulting in the evolution of flammable volatiles, and the gas phase pyrolysis or oxidation of the evolved gases. Chemical fire retardants may operate in the solid phase to modify the thermal degradation process or in the gas phase by yielding volatile products that inhibit the flame reactions. A given retardant formulation may, of course, operate in both modes.For purposes of discussion the general subject of fire chemistry may be usefully visualized in terms of the contributing phenomena of ignition, propagation, and cessation, as indicated by The detailed mechanism of solid-phase retardant action is a complex problem of organic reactions in the solid state. For the degradation of cellulose, for example, both free radical reactions and acid-catalyzed rearrangement pathways have been suggested. Speculations have been offered concerning the role of retardants such as phosphorus compounds in modifying the degradation . This asThe purpose of this paper, then, is to consider the molecular level mechanistic aspects of the gas-phase mode of fire retardancy. In dealing with this problem at the molecular level it will become apparent that there are fundamental connections in the details of the mechanisms underlying the phenomena of flame inhibition, fire extinction and smoke production. It is axiomatic that the question of toxic gas production is also related to these phenomena although this will not be pursued here.It is now recognized that the inhibition or extinguishment of flames can more effectively be achieved by chemical, rather than physical, means. However in practical fire retardancy situations, both physical and chemical effects operate. For the present discussion we will be concerned only with the chemical aspects of flame inhibition.A number of reviews and surveys, concerned primarily with the chemical aspects of flame inhibition, have been made –13. DiscIn practical systems the manner in which the additives are released to the flame, or potential flame, system is of importance. One of the theories based on empirical data requires the additive to be “at the right place at the right time.” Thus, to some extent the protection of flammable substrates requires that the vapor release properties of the inhibitor be related to the fuel release properties of the flammable substrate. As an example one may cite the antimony oxide — organic halogen formulations used to reduce the flammability of organic polymers where the antimony-halogen component is released at about the same time as the polymer decomposes .3 and HO2. The influence of additives on flame speed, also known as burning velocity, is particularly revealing in that only small amounts of additives are required to strongly affect flame propagation. It is also apparent that the inhibiting action of additives, such as halogens, cannot be explained merely in terms of a reduction of the equilibrium concentration of radicals due to hydrogen halide formation. Evidently the function of the additive is catalytic and flame inhibition is primarily a kinetic phenomenon  is the initial oxygen concentration,[OI] is the initial inhibitor concentration,[V0 is the burning velocity in the absence of any inhibitor, andV is the change in burning velocity due to the presence of the inhibitor.δFrom the mathematical model a figure of merit for an inhibitor, using burning velocity data, may be defined as:It is assumed that the flame is slightly fuel rich, i.e., the H atom is the dominant radical.vϕ correlates with other measures of inhibitor effectiveness, such as blow-off limits and extinction limits for halogen inhibitors. It also follows from the definition of vϕ that the degree of inhibition should be proportional to the amount of inhibitor added to the flame. Experimental observations , show tvϕ for a selected variety of inhibitor types have been calculated from the data of Lask et al. [n-hexane fueled flames, and D. Miller et al. [2 fueled flames. The values listed in the Values of k et al. , and unpk et al.  for n-her et al. , for H2 2 is the degree of inhibition explainable in terms of a physical effect such as cooling. The magnitude of vϕ greater than unity for the other inhibitors can only be explained in terms of chemical effects. Common commercial extinguishants such as Freons have vϕ values similar to the value of 8.4 given for Br2. Of the two flame types considered in the table the n-hexane/air system more closely resembles the chemical conditions likely to occur in practical fire situations.At this point it is sufficient to note that only for the case of COiX versus z, from which the derivatives are calculated.In order to provide a molecular basis for flame inhibition it is necessary to identify the elementary reactions involving the flame inhibition and flame propagating radicals. For the ideal case of a premixed laminar flow flat flame it is possible to determine reaction rates and in some cases identify elementary reaction steps. The basic flame relationships are given in 4 fueled flames are given in 4 as a portable source of fire extinguishment; (b) the current use of Freons such as CF3Br as an extinguishant particularly in connection with fuel fires associated with aircraft mishaps, and also for the protection of electronic equipment; and (c) the incorporation of phosphorus-halogen or antimony-halogen (among others) formulations in materials such as natural and synthetic fabrics, furniture materials, paints, wood, paper, etc., e.g., see Lyons [Halogen systems are among the most widely used commercial reagents for fire prevention and extinguishment. For example, one can cite (a) the early use of CCIee Lyons .Halogen systems have also received the most attention by workers interested in the basic mechanism of flame inhibition. It should be recognized that in some instances the halogen treatments serve to alter the course of decomposition of substrates such that only relatively nonflammable gases are produced during the pyrolysis or oxidation processes. However, the present discussion is restricted to the retardancy aspects that involve vapor phase chemistry in the inhibition process.In practical fire systems the halogen species can be introduced into the gas phase by mechanical means, as with Freon protection systems, or by chemical means, as with the release of HCl from decomposing Polyvinylchloride, or as phosphorus chlorides or oxychlorides formed during decomposition of a polymer substrate, or as antimony halides from polymer substrates.vϕ values given in 3Br is about four times more effective than CF4 in preventing combustion of n/hexane air mixtures; (c) CF3Br is a good inhibitor in hydrocarbon-air flames but is mediocre in H2-air flames; (d) Br2 has very little effect on CO—O2 flames but is effective when small amounts of H2 are introduced; and (e) in Br-substituted hydrocarbons and fluorocarbons the inhibitor effectiveness increases with the number of Br atom substitutions and, in certain instances, can be directly proportional to this number. For example, in a stoichiometric methane-air flame, the following vϕ values are found: Br2 [3Br [3Br [3Br seems anomalous and this has been attributed to a possible role of the CF3 radical in flame inhibition.As is evident from the high und: Br2 , CH3Br [Br2 [3Br , HBr [113Br [3Br . The higFrom these macroscopic observations it can be concluded that the flame inhibition must involve an interaction between species containing a halogen and a radical containing a H-atom, such as H or OH.A more detailed description of the likely flame inhibiting mechanism has been derived from experiments carried out at the molecular level, such as the mass spectrometric microprobe sampling experiments described by Wilson et al. , or by PBefore discussing the current status of the mechanistic understanding of halogen flame inhibition, it should be noted that a considerable variety of mechanisms have been proposed in recent years. For example, Mills  suggesteX may be CI or Br, as an intermediate is suggested; i.e.:Creitz  suggests3Br or HBr in hydrocarbon flames. The observed radiation was not from a very well defined part of the flame, both reaction zone and post flame regions being included. It is well known from flame spectrophotometry studies that emission intensity may not necessarily be correlated directly with radical concentration.Other workers have suggested that the halogens reduce the concentration of OH in the flame. Rosser et al.  observedIn time resolved studies on the low pressure explosive combustion of styrene-oxygen mixtures, Petrella  observed4—O2 flames containing HBr. However, using 1 atm lean propane fueled flames, Pownall and Simmons [The general situation with regard to the action of halogens on OH in flames remains unsettled, and some of the apparent difficulty may be related to the use of different flames for inhibition studies. In particular, Wilson et al.  have obt Simmons  provide Levy et al.  suggest f Wilson  that met-workers  have sug2 such as:4/O2 flame leads to the production of the hydrogen halide, HX, early in the flame. It was observed that the formation of H2CO is inhibited and the production of H2 enhanced. This provides indirect evidence for a removal of H atoms from the flame and the predominant reaction is considered to be:From the recent review of Fristrom and Sawyer , the curn et al. , it is ed Sawyer  have demy et al. ). The reThe production of HBr from other initial sources such as RBr can readily be achieved by reactions of the type:2 actually promotes flame propagation in H2-air flames, as shown by the negative vϕ given in Note that this inhibition mechanism readily accounts for the noninhibiting properties of fluorides, since the high stability of HF provides an excessively high activation energy barrier for a reaction with H atoms to result. The much lower effectiveness of chloride as compared with bromide inhibitors is probably due to the HCl reaction being very close to thermoneutral, and hence it is likely that the reaction can proceed in the back direction to generate H atoms. The probable importance of the back reaction is evident from the observation that ClThis seems to be a very satisfactory model for halogen flame inhibition although it does not account for all of the experimental observations . More r4 and BBr3. However SiCl4, TiCl4, CrO2Cl2 and Fe(CO)5 have no effect on the ignition temperature and CCl4 actually decreases the ignition temperature. Evidently the model is not of wide generality and it should be noted that the supporting flame microstructure evidence was obtained with 0.05 atm lean CH4-O2 flames.Another test of the model is provided by the ignition temperature studies of Morrison et al. . The modIn the higher temperature reaction zone region of flames, reactions involving H, OH, and O are known to be balanced and a pseudo-equilibrium exists between these species. Hence under these conditions, arguments as to whether OH, O, or H are the inhibited species are not particularly critical, as a reduction of any of these radicals would serve to inhibit chain branching.It is known that inhibition is more effective in cooler than in hotter, faster burning flames, and Friedman  has sugg2, are negligible compared to the observed chemical effects . The de3Br on the fuel side of diffusion flames (Creitz [It should be noted that the apparent low effectiveness of CF (Creitz ) is expl (Creitz  simply a3)3PO4, Pb(C2H3)4 and Fe(CO)5 are one to two orders of magnitude more effective than halogen inhibitors. However systems of this type have not yet been utilized in practice, except that lead tetraethyl has been used to modify the preignition knocking phenomena of internal combustion systems. It is not unreasonable to speculate that the modes of action in flame inhibition and knock prevention may be related. Unfortunately, despite considerable experimental effort, the mechanism of knock inhibition has not been definitively established.It is apparent from the classification of inhibitor effectiveness given in 2-air flame where Cl2 itself does not provide any flame inhibition. It is clear that the metals and phosphorus can themselves lead to flame inhibition and, more importantly, to a much greater degree than the halogen inhibitors. The two order of magnitude difference in vϕ between Si(CH3)4 and Pb(C2H5)4 suggests that the inhibition mechanism is particularly sensitive to the properties of the metal itself. In fact, some metals show no inhibition at all. For example, the addition of several percent of Al2Cl6 vapor to pre-mixed fuel-rich CH4/O2 flames produced a reduction in burning velocity that could be accounted for entirely from the amount of halogen present [The metal and phosphorus halides indicated in  present .5 reached a limiting upper concentration level. At the higher concentration levels, condensed particles should form and the decreased rate of effectiveness with composition implies that heterogeneous inhibition is less effective than homogeneous reactions. The question of heterogeneous versus homogeneous inhibition has received considerable attention in connection with the function of the commercially used solid extinguishants such as the alkali metal bicarbonates.Metals such as Fe, Cr, and Ti, and their oxides, have low vapor pressures under normal flame conditions and their introduction to flames can result in the formation of condensed particles. These particles are highly luminous and are readily observed in the region of the reaction zone and the post flame gases. It has been argued, therefore, that these metals perturb the flame chemistry via heterogeneous rather than homogeneous reactions. A clue to the possible effectiveness of heterogeneous, as opposed to homogeneous inhibition, is provided by the observation of Jost et al. , where t−3 mol fraction of powdered alkali metal salts such as K2SO4, Na2CO3, KHCO3, and NaHCO3 can reduce the flame speed of CH4-air flames by 50 percent. Thus vϕ factors of about 100 are indicated. Powders of alkali metal salts are also found to be more effective on a weight basis than CF3Br in a counter flow diffusion flame [It is found that about 10on flame . Their e3 on flame cooling is small and that the mode of inhibition must be chemical in nature. In general the smaller the initial particle size of the powder introduced to the flame, the greater the degree of inhibition found  show thg et al. ; and Rosg et al. ).2 gas retards the efficiency of the oxalate and cyanate. Also Friedman and Levy [From these observations, arguments have been given in favor of both solid and vapor phase inhibition mechanisms. Rosser et al.  favored and Levy  have obsMost workers agree that the active gas phase species would be the hydroxide, KOH. Under equilibrium conditions this is much more stable than the oxides or elemental K. Under very lean flame conditions the oxides may also be important (69). The reaction:15 cm−3 in H2-O2-N2 flames, particles were observed which showed a higher temperature than the flame gases, i.e.:The refractory nature of the other inhibiting metals and their oxides increases the likelihood of solid formation and a heterogeneous mode of inhibition. Radical recombinations on solid particles should lead to a temperature rise at the particle. For Cr concentrations greater than 10d Padley ).−5–10−6 mol fraction in a 1600 K H2-O2-N2 flame, solids do form prior to the reaction zone. These flames did not show the streakiness which is commonly observed when solid particles are present. However, in spite of the presence of solids the observed catalytic effect of alkaline earth metals on the recombination of H-atoms could be explained solely in terms of a homogeneous mechanism.In these studies observations were made downstream of the reaction zone and the question arises as to whether the additives have an opportunity to form solids in the preflame region or not. Very little has been done to answer such a question. However, from the light scattering experiments of Cotton and Jenkins , it is e4 flame containing Fe(CO)5 additions was reported by Vree et al., [Indirect evidence for the formation of solids in the pre-reaction zone region of a low pressure CH et al., .The post reaction zone radical recombination studies made in the presence of metal catalysts, represent the only molecular basis studies that have a bearing on the likely flame inhibiting function of metal containing species. The results of these studies have dispelled the often relied-upon notion that the presence of metallic elements at ppm concentration levels has no effect on flame chemistry.The survey experiments of Bulewicz and Padley , 70 as tvϕ values and they are also among the most effective catalysts for H-atom recombination. Hence it is reasonable to conclude that the role of the metallic flame inhibitors is somewhat analogous to that of the halogens, in that the overall effect results from catalysis of H-atom recombination.Many of these oxides and hydroxides are known to be stable under typical flame conditions. Unfortunately, only Sn and Cr are available for comparison with the flame speed measurements represented by 2 and ground state SnO. Such subtleties in energetics readily account for the stricking variance in the ability of metals to inhibit flames or at least catalyze radical recombination.It appears that the oxide-hydroxide mechanism relies on a rather special set of energetics. In particular, MOH must be stable under flame conditions but must readily react with a H-atom when the opportunity arises. Ideally this last step should be exothermic to reduce the probability of a reverse reaction. A more critical set of energetic conditions is revealed by the case of Sn , where t2 on the alkali metal flame inhibition can also be expected to occur for some of the other metal inhibitors. In particular, flame systems containing metals such as Ba, Cr, and Ca, and a halogen source can be expected to form stable MCl and MCl2 species. This would reduce the effectiveness of these metals, as they would not be available to participate in the MO—MOH inhibition sequence. On the other hand, metals such as Sb and Sn have relatively weak metal-halogen bonds and the halogen poisoning effect would be minimal in these cases.The poisoning effect of Cl3, to the vapor phase it was suggested that the main role of antimony was to moderate the release of halogen to the flame. However, there is evidence to suggest that, given the opportunity to enter the vapor phase, antimony, in the absence of a halogen, can provide a useful degree of flame inhibition. In particular, triphenylstibine shows all the characteristics of an effective vapor phase flame inhibitor [vϕ for SbCl3 as compared with Cl2 or CCl4  adjustment of the operational parameters such as fuel ratio and aerodynamic flow conditions as has been suggested for smoke reduction in jet engines ; (b) useAs was the case with flame inhibition, one is faced with the problem of setting up criteria for defining the degree of effectiveness of additives as smoke reducers Thus Cotton et al.  chose thThe smoke inhibition mechanism suggested by Cotton et al.  is parti2 as a carrier gas.The survey of Cotton et al. , involve−4–10−5 mol fraction.The efficiencies of the various metals, relative to Ba, as soot suppressants are represented in the The metals not shown in this figure had essentially no effect relative to Ba and they include Ag, Al, As, Be, Cd, Co, Cu, Fe, Mg, Ni, Pb, Si, Th, Ti, Tl, U, and Zn. Notably, several of these, namely Fe, Pb, Si, and Ti, were found to be very good flame inhibitors as discussed elsewhere. From the 2. From known rate data, the mechanism was demonstrated rather convincingly, at least at a semiquantitative level. Basically the model is as follows. Under the fuel rich conditions necessary for soot production, the active radical concentrations are low and in fact may even be below the equilibrium level. This follows from the well known self-inhibiting effect of hydrocarbons, i.e.,A common mechanism was proposed for the soot suppression activity of Ba, Sr, Ca, NO, and SOThe flame condition should be contrasted with that found in faster burning flames, where premixing and less fuel rich conditions exist, with the result that the reaction zone radical levels are well in excess of the equilibrium concentrations. Under these latter conditions Cotton et al. , found tThe presence of MO in flames, where M is Ba, Sr or Ca, is well established, as is the formation of the metal hydroxide intermediates. Also the balanced nature of reaction 4 is well established, at least for the hotter flame regions. Thus the net effect of these processes is to increase the formation of OH radicals and hence the oxidation of solid carbon. Note that the reaction 2 leading to the production of the metal dihydroxide is endothermic, and this suggests that the suppression of smoke will be much less effective as the temperature is decreased.From this model it can be argued that it is not possible to utilize metals for the simultaneous reduction of smoke and flame propagation. This is also suggested by the observation that, as the flames become increasingly more fuel-rich, the catalytic effect on radical recombination is substantially diminished.Also in real fires, the degree of fuel-oxidant mixing will be an important factor in determining the effectiveness of metallic inhibitors. From arguements such as these, it is not at all surprising that the effectiveness of fire retardant treatments may vary according to the test conditions used. It is also evident that the presence of solid carbon in flames has an inhibiting effect on flame propagation. We may recall that the use of halogens as flame inhibitors often involves an increase in smoke production. Thus smoke production and flame propagation are intimately related phenomena and the use of additives to exclusively suppress one of these, without affecting the other, does not appear to be possible.As much of the experimental evidence for the present understanding of flame inhibition has been derived from molecular studies on low pressure flames, it is important, for scaling purposes, to consider the effect of pressure on inhibition.5 decreased and at low pressure had little effect on the OH concentration or the flame temperature. Inhibition was found to be about a factor of three greater at 1 atm than at half an atmosphere. Similar results were obtained for the effect of trimethyl phosphate (TMP) on the flame inhibition chemistry of H2—O2—Ar flames [−3 mol fraction TMP; whereas at 1 atm pressure inhibition was observed. A factor of two reduction in the H-atom concentration was also observed when a 10−3 mol fraction of TMP was added to the 1 atm flames. Fenimore et al., [Bonne et al.  found thr flames . Flames  et al.,  suggeste5, CH3I, CH3Br, and CH2Br2 but not for CH2Cl2. In particular, the Fe(CO)5 inhibitor was the system most affected by a pressure change. The authors suggest the possibility that this effect is indicative of the role of termolecular reactions, in addition to the normally considered bimolecular steps, in the inhibition process. Such an argument is in keeping with the proposed model for metal oxide inhibition via the hydroxide intermediate.More recently, Homann and Poss  have fouFrom these few observations it is clear that one should proceed with caution in attempting to extrapolate the results of inhibition studies on low pressure flames to the “real-life” application of 1 atm systems.It is evident that inorganic systems containing halogens (other than F) or certain metals can provide a degree of flame inhibition which is considerably greater than that allowed by physical processes. It also appears that high temperature species such as metal halides, oxides and hydroxides play an important part in the flame inhibition process. As the flame inhibiting processes are kinetically controlled and rely rather stringently on a special set of energetics, the need for basic thermochemical and kinetic data, in support of flame inhibition mechanistic studies, is apparent. Rates of vaporization under pyrolysis and flame conditions also appear to be important quantities in the overall flame inhibition process.2-air flames [The basic feature of all of the flame inhibiting systems considered is their interaction with H-atoms as the mode of inhibition. In this connection it should be noted that hydrocarbons are themselves strong flame inhibitors. This is particularly evident when hydrocarbons are introduced into Hr flames . The inhFrom the flame speed studies of Miller et al.,  it is ev4 and SiCl4 it is most unlikely that any appreciable formation of the monoxide would occur prior to the reaction zone. For such low concentrations of metal additives it is not unreasonable that the onset of condensation and particle formation should lead to a reduction in the rate of inhibition with additive concentration. The formation of particulates would tend to lower the collision probability of radicals with the additive.It also appears that the inhibition of H atoms can be effectively achieved in both preflame and reaction zone flame regions. The most reasonable model for the inhibiting effect of metals is one involving participation of metal oxide and hydroxide species. This effect is believed to be important primarily in the region of the reaction zone. From the high metal-halogen bond energies for additives such as TiCl2 is actually accelerated by the presence of Cr at the ppm concentration level . A simi3 and PBr3 have the same effectiveness in n-hexane-air mixtures when Br2 and HBr are substantially superior to Cl2 or HCl? Why is the inhibiting action of halogens more pronounced for fuels with low hydrogen content  than for H2 or CH4 [5 much less effective in H2-fueled flames? Similarly, why is Fe(CO)5 less effective in an O2-CH4 flame than an air-CH4 stoichiometric flame [It is apparent that the various suggested mechanisms do not satisfy all of the macroscopic observations and general questions arise. For example, why do PCl2 or CH4 ? Why is ic flame ?Answers to such questions and a definitive understanding of the mechanisms involved in flame inhibition can only result from molecular level studies of flame reaction kinetics.


Acute lymphoid leukemia is the most common pediatric cancer. Several factors may be found associated with leukemic state and previous allergic disease needs to be evaluated.To investigate a possible association between preceding history of allergy disease and high Total IgE levels with Acute Lymphoid Leukemia in children and adolescents.A hospital-based case-control study in the northeast region of Brazil. The population included 60 patients diagnosed with non-T acute lymphoid leukemia by means of bone marrow myelogram and immunophenotyping and 120 controls selected proportionally for age and gender. Data collection employed a structured questionnaire to determine prior history of allergies, as well as total IgE serum tests and clinical evaluations.The results show that both total IgE serum levels and preceding history of asthma have a significant and inverse risk association with acute lymphoid leukemia.In the unadjusted model, the exposure variables with a p-value < 0.20 were: asthma, high IgE levels, concomitant allergic diseases, atopic dermatitis, allergic rhinitis and urticaria. The inverse relation to both asthma and IgE serum levels continued to be significant in the adjusted model, showing significant p-value 0.044 and OR (CI 95%) of 0.14 (0.02 – 0.95); and p-value 0.001 and OR (CI 95%) of 0.10 (0.02 – 0.41), respectively.High levels of total IgE, especially related to asthma, appear to contribute to alterations in the immune system, which may activate Th2-mediated pathways and this fact might confer some protective role against acute lymphoid leukemia.


Development of new cryopreservation strategies has major potential in medicine and agriculture and is critical to the conservation of endangered species that currently cannot be preserved. A critical property of any potential cryopreservative solution is its ability to prevent cell-damaging ice formation during cooling and subsequent heating. This study focuses on the freezing behaviour of promising model cryoprotective solutions. We perform neutron scattering analysis, combined with computer modelling, of the water structure after quench cooling these solutions. It is found that water in this solution forms nano-clusters encapsulated by the surrounding matrix of cryoprotectant solute molecules. We posit that these small volumes inhibit ice formation, because water does not have space for the structural relaxation required to crystallize on the timescale of the cooling process. The success of this process, known as ‘cryopreservation’ , dependsVitrification, a quench-cooling process which involves the sudden solidification of water-containing objects, may avoid the formation of either internal or external crystalline ice, and so is a promising alternative approach to cell preservation which is sometimes successful where more traditional freezing methods fail ,2,7. WhiCyprinus carpio) embryos by alcalase (proteolytic enzyme) treatment to increase the chorion permeability for cryoprotectants was examined. Two minutes incubation with alcalase was selected as the optimal time for the following experiments, which involved stepwise incubation for 9 min in Hanks’-based solutions containing 1,2-propanediol (PD) and methanol (Met) within the concentration ranges of 5.75–23 vol% and 4.25–17 vol%, respectively. The embryos were either frozen as is or incubated for a further 5 min in a Hanks’-based cryoprotective medium with 23 vol% PD, 17 vol% Met and containing either 20–30 vol% dimethylsulfoxide (DMSO) or 37–57 vol% Met. To explore the toxicity of the different cryoprotective solutions, embryos were transferred into incubators filled with fresh hatchery water. After 1 day of incubation, the hatching rate was determined as a ratio of the number of live, moving embryos to the total number of embryos in the incubators. For the most concentrated cryoprotectants, the hatching rate was in the range 7–40%, while for incubations in media with lower cryoprotectant concentrations, hatching rates were in the range 84–95%. In other words, even at the highest concentrations there was a significant survival probability following treatment with the cryoprotectant solutions.The cryoprotectant solutions used here were formulated during a previous study of fish (common carp) embryo cryopreservation . In thata, when the embryo suspended in a medium containing 15.41 vol% PD and 11.39 vol% Met was quench-cooled to liquid nitrogen temperature (77 K), both the cryoprotective media and embryos appeared opaque, signalling either ice formation, protein denaturation, or some other aggregation phenomenon on at least the length scales of visible light wavelengths in both the embryo and the surrounding solution. Increased concentrations of cryoprotectant  in the cryoprotective medium led to the appearance of a transparent solution medium but opaque embryo (figure 1b). Finally, the use of a cryopreservative medium containing 23 vol% PD, 17 vol% Met and 20 vol% DMSO retained transparency for both the medium and embryo when examined in the cold condition (figure 1c). While transparency by itself cannot be regarded as a direct indicator of vitrification over all length scales, the differences between the three cases are quite striking. Protein denaturation, some other aggregation phenomena or ice crystallization will almost certainly lead to a degree of opaqueness [For the work shown here, freezing was achieved using solid surface vitrification, by dropping the embryo onto a metal plate which had been pre-cooled to liquid nitrogen temperature. The use of this series of cryoprotective media with gradually increasing cryoprotectant concentration led to several variants of the sample response on cooling, three of which are shown in figure 1aqueness . Hence iBecause interpretation of the optical transparency of an object during cooling cannot be used as direct confirmation of the presence or the absence of ice or other heterogeneities, further study is needed. In particular, because microcrystals or other aggregates (of dimension ∼ nm) could potentially be on a scale which is not accessible to optical inspection, and because such heterogeneities might play a decisive role in cryopreservative behaviour, by initiating unstable and damaging ice growth to larger length scales, a specific probe of nano-scale structure is required. Total neutron scattering, with its ability to uncover atomic arrangements at the 0.01–10 nm length scale and to ‘see’ the hydrogen atoms in water and other molecules of the solution, can shed light on the extent of ice crystal formation during cooling, while at the same time giving detailed insight into the way the molecules in these cryoprotectant solutions are organized. This information is vital for understanding the vitrification process in complicated biological objects such as fish embryos, and has the potential to direct the search for better aqueous media for cryopreservation.A recent study has investigated the way one of these solutions behaves on quench cooling followed by gradual heating . General2.2.1We prepared 2 ml of each of the solutions described in 2.2partial, structure factors, Hαβ(Q), where α, β represent the pair of atom sites for each term, and Q is the wavevector change in the scattering experiment:cα the atomic fraction and bα the neutron scattering length for atom type α. Hαβ(Q) is the Fourier transform of the corresponding site–site radial distribution function [ρ is the total atomic number density in the system. In terms of these radial distribution functions, the coordination number of β type atoms around an α type atom at the origin, Nαβ(r), is defined by the integralrmax is typically the position of the minimum in gαβ(r) beyond the first peak.Scattering data like these represent a weighted sum over a set of site–site, or function :2.2Hαβ . This me3.We studied the molecular structure of the quenched aqueous mixtures which had been used in the fish embryo cryopreservation experiments described previously . As is sx-, y- and z-axes, making a total of order 1.0×106 pixels, with the pixel step size set to approximately 0.05 nm. The exact number of pixels used in this routine is optimized depending on the distance criteria being used to define a void. Each pixel is assigned a status of either ‘occupied’ or ‘not occupied’. To be classed as occupied, there must be at least one atom of a specified type within a specified distance from that pixel; otherwise, the pixel is classed as not occupied. In the present case, each water molecule oxygen atom was used to determine if a pixel was occupied or not. At the end, the array of occupied pixels is, to a good approximation, a representation of the space occupied by the water molecules in this solution.In order to characterize the shape of the space occupied by water molecules in this solution, we have performed a so-called ‘void’ analysis on the simulation box . In thisshape of this space an autocorrelation is performed on these occupied pixels, giving a radial distribution function of occupied pixels, otherwise called the ‘volume distribution function’, gV(r). Various texts from the theory of small-angle scattering have given functional forms for this correlation function depending on the shape of the space involved . The caed  in thisequation  are 0.034.inside the cell with a minimum of toxicity effects. According to The foregoing results provide some general pointers as to how to avoid nucleation of ice when cooling biological organisms, particularly those which are difficult or impossible to cryopreserve, such as fish embryos. However, given that bulk water crystallizes rapidly even when suddenly quenched to liquid nitrogen temperatures, the cooling rate requirement is only part of the requirement. Slowing the rate of crystallization to below what happens in the bulk requires there to be, in addition, an underlying matrix which is able to sufficiently confine the water into small volumes so that ice nucleation becomes impossible on the timescale of the cooling. Concentrated sorbitol–water mixtures apparently provide such a matrix , as doesc, survived heating when attempting to return the embryos to their pre-frozen state. The rate of cooling obviously has to be fast enough to prevent ice nucleation, but there is in addition the likelihood of ice formation on thawing [figure 1c). In that case, the embryo could apparently survive vitrification treatment (i.e. did not show ice formation with vitrification) with the present cryoprotective solution (samples 4–7), and this same solution also does not show ice formation when rapidly cooled to 77 K on its own. Other solutions that showed ice formation in the common carp embryos also showed ice formation when rapidly cooled on their own . However, how we might overcome the remaining ice nucleation obstacles associated with warming remains a project for future study.It must be emphasized that none of the embryos depicted in figure 1 thawing . Hence i


The CO2 vent found at Levante Bay  also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments—the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone’s tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO Recent  volume) . This unTrace elements are bioconcentrated in marine invertebrates by at least one order of magnitude when compared to seawater . Some trAnemonia viridis is a temperate Mediterranean species, which occurs naturally throughout Levante Bay. The anemones show no apparent signs of stress to the prevalent environmental conditions close to the primary vent  and animal tissues.The simultaneous exposure of 2 vents create a natural CO2/pH gradient along the north-easterly side of the bay ranging from pH 6.05 to 8.29 at >350 m from the vent site (pCO2) was in proximity to the CO2 vents (∼260 m) in accordance with previous studies , part of the Aeolian Island chain, NE Sicily  in May 2ent site . The venent site . Two sitent site ; Site 1  studies .A. viridis, a dominant benthic organism in Levante Bay, was prevalent throughout the study area  and then converted from lux to µmol quanta m−2 s−1  and temperature were measured several times a day for 4 days using a pH meter . Water samples for total alkalinity (TA) analyses were collected from each site, cooled and stored in the dark until analysis. TA was quantified with a Metrohm 862 compact titrosampler . The pCOsalinity , using tsalinity , selecti m−2 s−1 .MΩ; MQ Academic) and double distilled acids. All test tubes were trace-element clean certified (Stardest) and other lab ware was thoroughly cleaned with acids  for 24 h following a thorough rinse in DDW water). Solutions were prepared with DDW water and analytical grade or higher purity salts.Special care was taken to minimize sample contamination during handling. Samples were treated in a trace element clean facility  equipped with three class 100 laminar flow hoods, using double distilled water  and the pedal disc (n = 10 per site) of each anemone. The subsamples were placed in pre-weighed trace element clean 15 ml test-tubes and their wet weight recorded with a sensitive scale .To examine the distribution of trace elements in the anemone body, two distinct body compartments were excised and analyzed separately . Duplican = 5 per site). The subsamples were placed immediately in a trace element clean pre-weighed 15 ml test tube and their wet weight recorded. One duplicate was digested in acid to yield the total tentacle trace element concentration. The other duplicate received 5 ml NaCl solution (35 g L−l) and was homogenized for 3 min with an electric homogenizer . The homogenate was centrifuged for 5 min at 5,000 rpm  to separate the Symbiodinium (pellet) and the host tissue (supernatant). Visual inspections revealed no crossover of material between these components, but both were washed carefully. The host supernatant was homogenized and centrifuged for 10 min at 13,500 rpm. Then, the pelleted host material was washed three times by means of re-suspension in 3 ml NaCl solution (20 g L−l) and centrifuged for 5 min at 13,500 rpm. The Symbiodinium pellet was washed three times by means of re-suspension in 2 ml NaCl solution (20 g L−l) and centrifuged for 3 min at 5,000 rpm.To further examine the partitioning of trace elements between the symbiotic algae and anemone host tissue, duplicate subsamples of ∼1 gr were excised from the tentacles of each anemone  was carried out using an Agilent 7500cx ICP-MS (Agilent Inc.). A set of calibration standards was added to the analysis together with appropriate blanks. An internal standard solution containing Sc, Re and Rh was introduced during the analyses to all standards and samples. The reported values are blank subtracted and averaged over duplicate analysis, with difference in % between duplicate analyses typically smaller than 10%. Results are given in ng trace element per mg dry weight (wt) of anemone.Samples were digested within the test tubes in the presence of 5–10 mL double distilled 9.8 N HNOSymbiodinium and anemone tissue was analyzed using the Kruskal–Wallis test. Post-hoc Mann–Whitney U-tests were run for separation of significant factors. Differences between factors were considered significant for a p value <0.05. Unless otherwise specified, mean values are presented ± SEM. All analyses were performed using SPSS version 20 .Differences between pedal disc and tentacles in each site, and differences between sites for each body compartment were tested with one-way ANOVA and a multiple comparison test of means (Tukey). Prior to each analysis, all data were checked for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Cochran’s test. The effect of the site on trace element concentrations in tentacle −2), consistent with findings by pCO2 site, anemones appeared to be healthy with their tentacles fully extended and without any excess amounts of mucus  was strongly elevated compared to typical seawater conditions that were recorded at the control site (−2 s−1) were similar between sites (1–2 m depth), while TA was elevated at the high pCO2 site  next to the vent than at the control site (Next to the vent the pH was significantly lowered and the rol site . SeawateCO2 site . The susrol site  and S2. rol site . Macro eOur sampling scheme provided us with a paired data set of trace element concentrations (per unit of dry weight) in the pedal disc and the tentacles of each individual anemone. Plotting the concentrations of each of the 12 tested trace elements in the pedal disc vs. that in the tentacles (10 per site), we can visualize differences among sampling sites and body compartments, as well as variability among individual anemones  and S1. p < 0.05; p < 0.05; pCO2 site, all trace elements except Sr, were more concentrated in the pedal disc than in the tentacles , (2) Group II: Trace elements that are accumulated in the pedal disc but are slightly depleted in the tentacles at the high pCO2 site , and (3) Group III: Trace elements that are depleted in both body compartments at the high pCO2 site . Statistical differences between sites are found for most trace elements mentioned above (p < 0.05), except Co and Group II elements in the tentacles, and Cd and As in the pedal disc , whereas there was a significant decrease from 3,162 ± 168 to 2,201 ± 197 ng/mg dry wt in the tentacles (p < 0.05).Macro element concentrations  did not change in both body compartments between sites. P, however, significantly increased in the pedal disc at the high Having analyzed the algae and host fractions as well as the whole tentacle, we can probe for contamination or trace element loss in the separation procedure, by comparing the sum of the separated fractions to the whole tentacle . Six of the trace elements  were screened out due to contamination or detection problems. In three trace elements , that were mostly associated with the algae, the sum of the separated fractions balanced out adequately with the total. In the other trace elements , which were mostly associated with the host, the total greatly exceeded the sum of the separated fractions, indicative of significant metal loss. Hence, we chose not to include the measured host fraction. Instead, it was calculated from the difference between the total and algal fraction ; Table 3p < 0.05 for whole tentacle trace element concentrations; p < 0.05). Cd, however, was lost predominantly from the host tissue and the low remaining Cd was evenly distributed between the algae (45%) and host tissue (55%)  .2 vents at Levante Bay of Vulcano Island are intensively studied within the frame of OA research. Given the intrinsic geochemical complexity of the location, we studied the combined effects of trace element exposure and elevated pCO2 on the anemone A. viridis. The present study provides important insights into the performance of A. viridis under natural (control) and enriched trace element conditions. Collectively, our data shows that trace elements are differentially accumulated in A. viridis between ambient (control) and low pH/high pCO2 environments, which confirms our hypothesis . The trace element accumulation pathways consist of: absorbance across the body surface, ingestion of particles, and/or active predation  increased in both body compartments at the high Group I; , while fII; pCO2  may contII; pCO2 , which dII; pCO2 . It is cII; pCO2 . Our meaII; pCO2  and S2.pCO2 site in our study) is responsible for enhanced Fe solubility of 40%, causing an increase in Fe bioavailability in the water column. Accordingly, a high seawater concentration of dissolved Fe was reported close to the primary CO2 vent , although there was enhanced accumulation in the leaves . Similar to Cd and Zn, As is associated with the host tissue  light intensities at stations 1 (control) and 2 (high pCO2). Both sites were at 1–2 m depth. Data represent the mean value (± SD).Measurements of pH, TA, temperature, calculated Click here for additional data file.10.7717/peerj.538/supp-2Table S2A. viridis. Plankton hand nets  were towed at each site for 15 min tocollect plankton and other suspended particulate material from seawater. The measured concentrations (ppb) are presented per tow.Suspended particulate material was sampled to assess the trace element content in the potential prey of Click here for additional data file.10.7717/peerj.538/supp-3Figure S1Ten anemones were sampled from each site and analyzed for trace element concentrations (ng/mg dry wt) in the tentacles and pedal disc. (A) Manganese, (B) barium, (C) nickel, and (D) chromium.Click here for additional data file.


P. vivax exposures provide strain-transcending immunity and whether their presence is associated with DBPII haplotypes found in patients with acute P. vivax. The ability of antibodies to inhibit DBL-TH-erythrocyte binding was tested by COS7 erythrocyte binding inhibition assay. Seven samples of high responders (HR) were identified from screening anti-DBPII levels. HR no.3 and HR no.6 highly inhibited all DBL-TH binding to erythrocytes, by >80%. Antibodies from these two patients’ plasma had the potential to be broadly inhibitory against DBL-TH1, -TH2, -TH6, -TH7, -TH8 and -TH9 haplotypes when plasma was serially diluted from 1:500 to 1:2000. To further examine the association of DBPII haplotypes and the ability of antibodies to broadly inhibit DBL-TH variants, the individual samples underwent sequencing analysis and the inhibitory function of the anti-DBPII antibodies was tested. The patterns of DBPII polymorphisms in acute patients were classified into two groups, DBPII Sal I (55%) and DBL-TH variants (45%). Plasma from Sal I and DBPII-TH patients who had the highest inhibition against Sal I or DBL-TH4 and -TH5 was serially diluted from 1:500 to 1:2000 and their inhibitory capacity was tested against a panel of DBL-TH haplotypes. Results provided evidence of both strain-transcending inhibition as well as strain-specific inhibition by antibodies that blocked erythrocyte binding against some DBL-TH variants and against homologous alleles. This study demonstrated broad inhibition by anti-DBPII antibodies against DBL-TH haplotypes in natural P. vivax exposed individuals. The identification of conserved epitopes among DBL-TH may have implications for vaccine development of a DBPII-based vaccine against diverse P. vivax infections.Duffy binding protein region II (DBPII) is a promising vaccine candidate against vivax malaria. However, polymorphisms of DBPII are the major obstacle to designing a successful vaccine. Here, we examined whether anti-DBPII antibodies from individual  The malaria asexual cycle involves repeated cycles of parasite growth and subsequent destruction of host erythrocytes. Each cycle is characterized by production of merozoites which recognize and invade new erythrocytes for continuation of the parasite cycle. The clinical manifestations of malaria are associated with asexual erythrocytic stages of the parasites . TherefoPlasmodium vivax Duffy Binding Protein (PvDBP) is released from micronemes during initial attachment of merozoites to the erythrocytes and for junction formation to complete the invasion process. The critical binding motif of PvDBP is referred to as DBP region II (DBPII) or the DBL domain. The interaction between DBPII and its cognate receptor, the Duffy antigen/receptor for chemokine (DARC) on the reticulocyte surface is required for parasite invasion  = 0.25 ± 0.08, naive controls, OD = 0.13 ± 0.030, P < 0.05, To evaluate inhibitory response against DBL-TH haplotypes in high anti-DBPII responders, the reactivity of naturally acquired antibodies in vivax patients (n = 103) was tested against recombinant DBPII protein by ELISA. The anti-DBPII antibody levels in acute human plasma were significantly higher than naïve controls  and 18 (45%) patients infected with DBP Sal I strain and DBL-TH haplotypes, respectively. For patients infected with DBL-TH haplotypes, 12 haplotypes were defined among P. vivax isolates. Nine haplotypes  were similar to our previously reported haplotypes [To further evaluate the association of DBPII polymorphisms and inhibition activity of anti-DBPII antibodies in Since the mutation of amino acids has been shown to change antigenic character of DBPII , in thisP. vivax infection, plasma samples from acute P.vivax patients no.9 and no.15 who had the highest broadly inhibition activity against heterologous DBL-TH4, DBL-TH5 and reference Sal I binding were used to evaluate inhibition against a panel of DBL-TH haplotypes by COS7 cell binding-inhibition assay after serial dilution from 1:500 to 1:2000.To support the ability of anti-DBPII antibodies in inhibitory response against both homologous and heterologous during P > 0.05). However, statistical analysis demonstrated significantly different inhibitory activity against DBL-TH1 when plasma was diluted at 1:1000 or 1:2000 (P < 0.05). In contrast, inhibition activity against DBL-TH binding of patient no.15 was not strain-transcending. There was significantly different inhibition against 8 DBL-TH haplotypes and the Sal I haplotype binding to human erythrocyte when plasma was serially diluted from 1:500 to 1:2000 (P < 0.05).For patient no.9, the inhibition capacity typified strain-transcending inhibition showing broad inhibition against heterologous DBL-TH2, -TH4, -TH5, -TH6, -TH7, -TH8, -TH9 and homologous Sal I strain . PercentP > 0.05). However, this patient had a low inhibitory immune response to heterologous DBL-TH1, -TH4, -TH5, TH-8, -TH9 and Sal I strain at 1:500, 1:1000 and 1:2000 plasma dilutions (P > 0.05) whereas inhibition activity against homologous DBL-TH4 and heterologous -TH5, -TH9-erythrocyte binding was very low at 1:500, 1:1000 and 1:2000 plasma dilutions . However, the inhibition activity against heterologous DBL-TH1, -TH4, -TH5 and Sal I was significantly different among DBL-TH haplotypes at 1:500, 1:1000 and 1:2000. These data suggest that patients infected with the new DBL-TH variant produced neutralizing antibody specific to conserved epitopes of DBL-TH2, -TH6, -TH7, -TH8 and -TH9 (P < 0.05), but strain-specific antibodies towards DBL-TH1, -TH4, -TH5 and Sal I  and Apical Membrane Antigen 1 (AMA-1) are highly polymorphic surface proteins that stimulated strain-specific immunity [dbpII alleles and the immunological response to DBPII variant antigens may help in designing vaccines. Therefore, the purpose of this study was to evaluate whether sequence polymorphisms in dbpII genes affect the inhibitory function of naturally occurring antibody to eight sequence variants of DBL-TH antigens , which may have implications for development of DBPII-based vaccine.Developing a vaccine for immunity , 15, 16.immunity , 18. Theimmunity , 20. Recimmunity . UnderstdbpII alleles among P. vivax isolates has been reported from different geographical regions, including Brazil [The analysis of genetic diversity of g Brazil , Colombig Brazil , South Kg Brazil , Thailang Brazil , Sri Lang Brazil , Iran [2g Brazil , Myanmarg Brazil  and Papug Brazil , 29. Amog Brazil –30. Althg Brazil , 10, 13.g Brazil . I503K hg Brazil . TherefoP. vivax infection, the inhibitory function of naturally acquired anti-DBPII antibodies against a panel DBL-TH variants was evaluated in acutely infected P. vivax patients infected with Sal I or DBL-TH haplotypes. The result demonstrated that patients who were infected with P. vivax expressing the DBP Sal I allele showed a high prevalence of strain-transcending inhibition towards DBL-TH strains with 50.0% and 40.1% of Sal I patients having broad inhibition against DBL-TH4 and DBL-TH5 haplotypes, respectively, whereas only 13.82% of patients produced antibodies to inhibit homologous Sal I DBP. Additionally, most vivax patients infected with DBL-TH variants had inhibitory antibodies against heterologous DBL-TH4 (33.33%) or DBL-TH5 (27.78%) whereas patients had no inhibitory immune response against heterologous Sal I stain. These data suggested that vivax patients infected with Sal I strain could produce broadly neutralizing antibodies to heterologous dbpII Thai variant alleles. However, the patients infected with DBL-TH variants rarely produced broadly neutralizing antibodies against reference Sal I stain. Interestingly, some vivax patients infected with DBL-TH variants had anti-DBPII antibodies to inhibit both homologous and heterologous DBL-TH variants. Together, this study support that DBPII variation is an evasion mechanism responsible for strain-specific immunity and that stable broadly neutralizing antibodies are achieved when antibodies target functionally conserved epitopes.To understand protective immunity against DBL-TH haplotypes in individuals exposed to P. vivax infection [Ntumngia et al [dbpII alleles including DBPII Thai haplotypes [P. vivax endemic areas. Here, the association of DBPII Thai polymorphisms with broad inhibition of anti-DBPII antibodies was examined in acutely infected P. vivax patients. Among vivax patients infected with reference Sal I strain, patient no.9 produced broadly neutralizing antibody blocking heterologous DBL-TH and Sal I-erythrocyte binding (P < 0.05). Among vivax patients infected with DBPII Thai variants, patient no.25, infected with DBL-TH2, produced neutralizing antibody against homologous DBL-TH2 and heterologous DBL-TH6 and -TH7 . The participant information sheet was written and approved by Committee on Human Rights Related to Human Experimentation, Mahidol University, and the Ministry of Health, Thailand. The informed consent was signed by each participant before the blood sample was collected. The selecting criteria of the patients were as followings: (1) systolic blood pressure not less than 90 mm, (2) body temperature not higher than 40°C, (3) hematocrit not less than 25% and (4) age of 18 or above. Those who did not fit the criteria were excluded. The minority participants were not involved in the study.P. vivax patients at malaria clinics in Chumphon province, which is in the Southern part of Thailand. The areas are malaria endemic with a high prevalence of P. vivax infections. One hundred three sera samples were collected in 2011–2012 from P. vivax patients for screening for anti-DBPII responses and evaluation of strain-transcending inhibition against DBL-TH variants. Forty blood samples were collected in 2014 to study the association of DBL-TH polymorphisms and broadly inhibitory activity of anti-DBPII antibodies in individuals with P. vivax exposure. Individuals were assigned for DBPII sequence analysis and testing of inhibitory function of antibodies against Thai DBPII haplotypes. Three blood spots were collected on filter paper from each consenting P. vivax patient for preparation of parasite isolates. Parasite genomic DNA was extracted with a QIAamp DNA mini kit . The confirmation of P. vivax infection was performed by microscopic examination of thin and thick Giemsa-stained blood smears. Blood samples for malaria-naive controls were obtained from 35 healthy volunteers who live in Bangkok and had no history of exposure to Plasmodium parasites. Acute P. vivax-infected volunteers who registered at Malarial Clinics and naïve control subjects were asked for informed consent under the protocol approved by the Ethic Committee on Human Rights Related to Human Experimentation, Mahidol University (MUIRB2012/079.2408).We collected blood samples from acutely infected Serological response against recombinant DBP reference Sal I was quantified by ELISA. Recombinant DBP region II (rDBPII) was expressed as a glutathione S-transferase (GST) fusion protein in E. coli. It was then affinity purified on glutathione and cleaved from GST with thrombin using standard methods . PurifieP. vivax patients, 40 blood spot samples were taken for DNA isolation and amplification. DBPII genes were PCR amplified as described in detail previously [To identify the DBP region II genotype of the causative agent in acutely infected eviously . In brieP. vivax patient to inhibit binding of DBL-TH variant and reference Sal I haplotypes to human erythrocytes. Expression plasmid constructs were engineered to express DBL-TH or reference Sal I alleles on the surface of transiently transfected COS-7 cells as fusion proteins to the N-terminus of enhanced green fluorescent protein (EGFP) [4 cells/well) in Dulbecco’s Modified Eagle Medium  with 10% fetal bovine serum . Recombinant plasmid DNA (100 ng/well) was mixed with Lipofectamine (2% Lipofectamine 2000/well) in DMEM without serum and then added into transfected wells (100 μl/well) and incubated in a humidified incubator with 5% CO2 at 37°C for 42–44 hr. The detection of the C-terminal green fluorescent protein (GFP)-expressing vector was used as positive control for checking transfection efficiency.COS7 cell erythrocyte binding assays were carried out to evaluate the ability of neutralizing antibodies in The inhibition of anti-DBPII neutralizing antibodies against DBPII-erythrocyte binding was performed as previously reported , 34. BriP-values < 0.05 were considered significant. The statistical analysis was performed and graphs prepared using GraphPad Prism .Comparison of anti-DBPII antibody levels between unpaired groups (patients compared to naive controls) was performed using the Mann-Whitney U test. The inhibition activity for each plasma sample was compared between all the DBL-TH alleles and tested for any statistically significant differences in antibody reactivity and inhibitory responses by one-way analysis of variance and multiple comparison analysis by Bonferroni test. S1 TableP. vivax patients (n = 40) Blood spot samples were taken for DNA isolation and amplification. DBPII genes were PCR amplified and PCR products were subsequently sequenced. The alignment of complete sequences of PvDBPII genes from 40 isolates were analyzed by CLUSTAL and percent similarity was assessed using BioEdit software.Data are presented DBL-TH haplotypes in acutely infected (DOCX)Click here for additional data file.