Quirijn De Mast
email: q.demast@aig.umcn.nl

Philip G De Groot

Waander L Van Heerde

Meta Roestenberg

Jeroen F Van Velzen

Bert Verbruggen

Mark Roest

Matthew Mccall

De Mast

An-Emmie Nieman

Josien Westendorp

Din Syafruddin

Rob Fijnheer

Edmee C Van Dongen-Lases

Robert W Sauerwein

Andre J Van Der Ven

Thrombocytopenia in

Keywords: PLATELET ACTIVATION, MALARIA, GLYCOPROTEIN-1B, VWF, THROMBOCYTOPENIA, PLATELETS Platelets, Thrombocytopenia, Platelet activation, Glycoprotein-1b, Malaria, VWF

Introduction

Thrombocytopenia is a common finding in patients with Plasmodium falciparum and Plasmodium vivax malaria. Apart from their central role in haemostasis, platelets contain a wide range of inflammatory, immune modulating and angiogenic factors [START_REF] Smyth | Platelet functions beyond hemostasis[END_REF].

Two recent studies have suggested that platelets play a distinct role in the innate host defense in the early stages of a malaria infection. McMorran and co-workers showed that purified human platelets were able to kill P. falciparum parasites within erythrocytes in an in vitro culture system, an effect that was dependent on platelet activation by adenosine 5'diphosphate (ADP) [START_REF] Mcmorran | Platelets kill intraerythrocytic malarial parasites and mediate survival to infection[END_REF]. Moreover, they showed that platelet deficient mice were more susceptible to death during infection with the murine malaria species P. chabaudi. Srivastava and co-workers showed that erythrocytes infected with the murine malaria parasite P. berghei interacted with and activated platelets, which resulted in secretion of CXCL4 (platelet factor 4), a platelet derived chemokine thought to be involved in the development of cerebral malaria [START_REF] Srivastava | Platelet factor 4 mediates inflammation in experimental cerebral malaria[END_REF].

So, platelet activation may be an important process in early malaria that contributes to the fall in platelet number and it raises concern over the use of aspirin as antipyretic in children with malaria [START_REF] Greenbaum | Platelets, pyrexia, and plasmodia[END_REF][START_REF] Pleass | Platelet power: sticky problems for sticky parasites?[END_REF]. However, to what extent platelets are activated during human malaria is uncertain. To our knowledge, there are no studies on platelet activation in early malaria, while the available data from patients with a more advanced malaria infection have been inconclusive [START_REF] Lee | Plasma macrophage colony-stimulating factor and P-selectin levels in malaria-associated thrombocytopenia[END_REF][START_REF] Supanaranond | In-vivo platelet activation and anomalous thrombospondin levels in severe falciparum malaria[END_REF]. There are also fundamental differences in the pathogenesis of human and murine malaria and caution should be taken when extrapolating findings from murine malaria models to human malaria [START_REF] White | The murine cerebral malaria phenomenon[END_REF].

Previously, we have reported a significant decrease in circulating platelet numbers in volunteers experimentally infected with P. falciparum during the earliest stages of the blood stage infection and we have shown that this decrease coincided with the onset of endothelial cell activation with release of the platelet binding protein von Willebrand factor (VWF) (de [START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF]. In the current study, we studied the occurrence of platelet activation, coagulopathy and reduced thrombopoiesis in this experimental human malaria model.

Observations made in these volunteers were confirmed in patients with naturally acquired malaria in Indonesia and in ex vivo studies

Design and methods

Experimental human P. falciparum infections

Design. Experimental human malaria infections with healthy and malaria naïve volunteers are invaluable for testing the efficacy of candidate malaria vaccines and a well established and highly reproducible model for studying pathogenic mechanisms in early malaria [START_REF] Epstein | Safety and clinical outcome of experimental challenge of human volunteers with Plasmodium falciparum-infected mosquitoes: an update[END_REF][START_REF] Verhage | Clinical outcome of experimental human malaria induced by Plasmodium falciparum-infected mosquitoes[END_REF]. The present study contains data from three experimental human malaria infections, which were carried out in 2005, 2007 and 2008. Details on the objectives, the characteristics of the volunteers and the clinical and parasitologic course have been described in detail before [START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF]de Mast et al, 2009b;[START_REF] Roestenberg | Protection against a malaria challenge by sporozoite inoculation[END_REF][START_REF] Mccall | Plasmodium falciparum infection causes proinflammatory priming of human TLR responses[END_REF]. The primary objective of the study in 2005 was to study immunological responses in early P. falciparum malaria [START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF][START_REF] Mccall | Plasmodium falciparum infection causes proinflammatory priming of human TLR responses[END_REF]; the latter two were intervention studies, assessing the protective efficacy of repetitive exposure to live malaria sporozoites under chloroquine prophylaxis [START_REF] Roestenberg | Protection against a malaria challenge by sporozoite inoculation[END_REF] and of a candidate malaria vaccine (study in 2008). The current study on platelet kinetics was among the predefined exploratory objectives of all three studies. Only volunteers belonging to the control groups of these intervention studies were included to avoid a confounding effect of the intervention. As previously described, all volunteers were infected by 5 P. falciparum infected mosquitoes and followed by thick blood smears and assessment of malaria symptoms three times daily until start of antimalarial treatment [START_REF] Verhage | Clinical outcome of experimental human malaria induced by Plasmodium falciparum-infected mosquitoes[END_REF]. Antimalarial treatment was started upon microscopic detection of parasites in a thick blood smear and consisted of a standard, 6 dose curative regimen of artemether/lumefantrine 80/480 mg (Riamet®), given at 0, 8, 24, 36, 48 and 60 hours. The presence of P. falciparum was also analyzed by PCR and this revealed that submicroscopic parasitemia is usually present 3 days before thick smears become positive and treatment is given. Five volunteers who did not develop a positive malaria blood smear and thus no thrombocytopenia and who received artemether-lumefantrine at day 21, according to the study protocol, were included as controls in some experiments. The Institutional Review Board of the Radboud University Nijmegen Medical Centre and the Dutch Central Committee on Research involving Human Subjects approved the study protocols (CMO 2004/129, CMO 2006/207 andNL14715.000.06, respectively), including these supplementary platelet studies. All volunteers gave written informed consent.

Sample collection.

Venous blood was collected daily between 08:00 and 10:00 AM in EDTA and in CTAD plasma tubes (Beckton Dickinson Vacutainer Systems, Europe). The latter contain the anticoagulant sodium citrate and the platelet stabilizing agents theophylline, adenosine and dipyrimadol. The CTAD tubes were kept on ice pending whole blood flow cytometric analyses or they were immediately centrifuged at 3500 rpm for 10 minutes followed by a second high speed centrifugation of the plasma to obtain platelet poor plasma for measurement of platelet circulating plasma markers. Plasma was stored at -80 °C awaiting analyses. Hemocytometry was performed on a Sysmex XE-5000 automated hematology analyzer (Sysmex Corporation, Kobe, Japan) within four hours after blood collection. Platelet production was assessed by measurement of the immature platelet fraction (IPF), a parameter for young platelets that was recently introduced on Sysmex analyzers [START_REF] Abe | A simple technique to determine thrombopoiesis level using immature platelet fraction (IPF)[END_REF].

Quantification of the IPF was performed after addition of a polymethine dye and making use of specialized software as described in detail previously [START_REF] Abe | A simple technique to determine thrombopoiesis level using immature platelet fraction (IPF)[END_REF]. The IPF is expressed as percentage of the total platelet number.

For measuring platelet activation by flow cytometry, whole blood in CTAD tubes was fixed with paraformaldehyde 1%, diluted with HEPES buffer and stained for 15 minutes with saturating concentrations of anti-CD41-PE, anti-CD62P-PE, PAC-1-FITC and anti-CD63-FITC. Data on 10,000 platelets in a log side-scatter versus log CD41-PE dot plot were acquired. The mean fluorescence intensity (given in arbitrary units) of each antibody was analyzed and corrected for non-specific binding of isotype control.

Circulating concentrations of the platelet products P-selectin, CXCL4 (platelet factor-4), CXCL7 (NAP-2), CCL5 (RANTES), CD40 ligand (CD40L) and soluble glycoprotein-1b (sGP1b; glycocalicin) were determined by using semiautomated ELISA on a TECAN RSP150 robot as previously reported in detail [START_REF] Snoep | High platelet reactivity is associated with myocardial infarction in premenopausal women: a population-based case-control study[END_REF]. Commercially available antibody duosets Dy137, Dy795, Dy393 and Dy278 (all R&D Systems, Wiesbaden, Germany) and antibody combination BMS239 (Bender Medsystems, Vienna, Austria) were used to determine concentrations of P-selectin, CXCL4, CXCL7, CCL5 and CD40L, respectively. Soluble glycoprotein-1b (sGP1b; glycocalicin) was determined by ELISA with an in house capture antibody (RUU-630) and a commercial detection antibody (M1852, Sanquin, Amsterdam, the Netherlands). Western blot analysis showed that this capture antibody is very specific for GP1b (data not shown). Intra-and inter-assay CV's of this assay were 4% and < 10%, respectively. Plasma thrombopoietin (TPO) concentrations were determined by a commercially available ELISA kit (R&D Systems, Wiesbaden, Germany). VWF and the VWF activation factor, which reflects the amount of VWF that is in a platelet binding conformation, was determined by ELISA as previously described [START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF]. Levels of D-dimer were measured by means of the STA Liatest D-Dimer and fibrinogen assay (Roche Diagnostics SpA, Mannheim, Germany). Activated partial thromboplastin time (APTT) was determined using a KC10A coagulometer (Amelung, Lemgo, Germany). Plasma (50 µl) was incubated for 2 minutes with 50 µl Patromtin (Behringwerke, Marburg, Germany). Coagulation was started with 50 µl of 25 µmol/l calcium chloride and the time to coagulation was measured.

Field study in Indonesia

Data obtained in the experimental human malaria studies were confirmed in patients with naturally acquired P. falciparum and P. vivax malaria, who presented to the Rumah Sakit Karitas Hospital in Weetabula, West Sumba, East Nusa Tenggara Province, Indonesia.

Consecutive subjects presenting to hospital with clinical symptoms of malaria and a P. falciparum or P. vivax parasite density of at least 2500 and 500 parasites/ µL, respectively, were enrolled. A group of healthy hospital staff was enrolled as controls. Characteristics of the malaria patients and the controls have been described in detail previously (de Mast et al, 2009a).

Ex vivo studies

The findings from the in vivo studies were further confirmed by in vitro studies. The effect of parasitized red blood cells (pRBCs) and various Toll like receptors (TLR) agonists on platelet activation status and platelet function were examined using flow cytometry and platelet aggregometry. Citrated blood (final concentration citrate 11mM) from two healthy volunteers was used for all experiments. P. falciparum asexual stages of the NF54 strain were cultured in vitro as described previously [START_REF] Mccall | Plasmodium falciparum infection causes proinflammatory priming of human TLR responses[END_REF]. Asynchronous cultures of NF54 strain parasites were harvested at a parasitemia of 10-20% and the mature asexual stages were purified by centrifugation on a 63% Percoll density gradient [START_REF] Mccall | Plasmodium falciparum infection causes proinflammatory priming of human TLR responses[END_REF]. This purification step results in preparations with 80-90% parasitemia, consisting of >95% schizonts/mature trophozoites. These preparations of pRBC were washed twice in RPMI 1640 and used fresh in stimulation assays or freeze-thawed twice to obtain parasite lysate. Mockcultured uninfected erythrocytes (uRBC) were obtained similarly and served as control.

Whole blood was also stimulated with the following TLR agonists: TLR2 with Pam3Cys (10 µg/ml; InvivoGen), TLR4 with purified Escherichia coli LPS (10 ng/ml), TLR9 with CpG ODN repeat (10 µg/ml; TIB MolBiol).

Flow cytometry. Citrate whole blood (90µl) was incubated for 30 minutes at 37 0 C with asynchronous pRBCs (final parasitemia ~5%), synchronized mature pRBCs (final parasitemia ~0.25%), uRBCs, lysate of synchronized pRBCs and uRBCs (2.5x10 6 pRBCs) or phosphate buffered saline (PBS). Further platelet activation was inhibited by the fibrin polymerization inhibitor Gly-Pro-Arg-Pro peptide (GPRP; Biotrend Chemicals AG, Zurich, Switzerland).

After adding saturating concentrations of antibodies, samples were incubated for 15 minutes in the dark at room temperature, followed by fixing with paraformaldehyde 10% for 10 minutes. The following antibodies were used: CD62P PECy5 (AK-4 clone; BD Pharmingen,

Statistical analysis

Data are presented as mean with standard deviation unless otherwise stated. Changes from baseline in serial data were analyzed with the use of a linear mixed model for repeated measures. No assumption about the shape of the time trend was made i.e. time point was included as a categorical variable. Correlation between repeated measures within subjects was assumed to only depend on the time between the repeated measures (Toeplitz correlation structure). Holm's method was used to adjust for multiple comparisons at several time points

Results

Experimental human malaria infections

Platelet kinetics. Platelet kinetics were studied in 11 volunteers. Parasitemia induced an early and marked decrease in platelet count from a mean value of 253 x 109/L to a mean nadir value of 113 x 10 9 /L (range 55 -188 x 10 9 /L) with a concurrent decrease in circulating leukocyte numbers (Fig 1A-B). In the early phase of the malaria infection, the IPF remained stable with a subsequent rise in the first days after the positive malaria blood smear, suggesting that the thrombopoietic capacity of the bone marrow was not suppressed ( 11 reference limit (0.5 µg/ml) in 9 out of 11 volunteers with a mean value of 2.6 µg/ml (range 0.2 -7.5 µg/ml). The mean fibrinogen concentration was 3.3 g/l (range 2.4 -4.4 g/l), and none of the volunteers had fibrinogen concentrations below the lower limit of the reference value (2.0 -4.0 g/l). Compared with the corresponding baseline values, there were no relevant changes (≥5 seconds) in the APTT in 9 of the 11 volunteers. A prolongation in APTT of 9.3 and 19 seconds was found in the remaining 2 volunteers with the latter occurring in the volunteer with the highest D-dimers value (7.5 µg/ml).

Increased sGP1b concentrations in absence of systemic platelet activation. Platelet activation status was measured at four pre-specified time points by whole blood flow cytometry in 15 volunteers (Fig 2 ). A slightly higher PAC-1 (αIIbβ3 activation marker) surface expression was found in some volunteers, but there was no concurrent increase in expression of the αgranule membrane protein CD62P (P-selectin) or the lysosomal marker CD63.

Since activated, adherent platelets may be missed with flow cytometry, plasma concentrations of a range of platelet membrane and granule products were measured in five volunteers. No consistent increase occurred in plasma concentrations of soluble P-selectin, CXCL4, CXCL7 and RANTES during parasitemia and its treatment (Fig 3A -D). Plasma CD40L concentrations were below the assay's lowest standard value in all samples. One volunteer had a transient increase in soluble P-selectin concentration, while RANTES concentrations were transiently increased in two others. In contrast, parasitemia and its treatment resulted in a significant increase in sGP1b concentrations in all volunteers from a mean concentration of 486.6 ng/ml at baseline to 704.2 ng/ml (p=0.04) at day 2 after treatment initiation (

sGP1b in naturally acquired malaria

To confirm our findings of a malaria-associated increase in sGP1b, we compared plasma concentrations of this protein adjusted to the individual platelet number between patients with symptomatic P. falciparum or P. vivax malaria and healthy controls on the Indonesian island Sumba. Patients with symptomatic P. falciparum and P. vivax malaria had significantly higher sGP1b concentrations per platelet than healthy controls (Fig 4A). Total sGP1b concentrations were also significantly higher in the falciparum and vivax malaria groups than in controls with median (IQR) values of 1074 ng/ml (696 -1511 ng/ml), 709 ng/ml (551 -1061 ng/ml) and 512 ng/ml (420-612 ng/ml), respectively. Recently, it was reported that shear-induced interaction of platelets with VWF results in GPIb-ectodomain shedding [START_REF] Cheng | Shear-induced interaction of platelets with von Willebrand factor results in glycoprotein Ibalpha shedding[END_REF].

In these Indonesian malaria patients, there was indeed a significant correlation between Platelets express various TLRs and because P. falciparum contains TLR4 and TLR9 ligands, these receptors may be involved in the presumed role of platelets in the innate immune response [START_REF] Aslam | Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in vivo[END_REF][START_REF] Cognasse | Evidence of Toll-like receptor molecules on human platelets[END_REF]. However, there was no up-or downregulated expression of the platelet receptors CD36, CD40 and TLR2, 4 and 9 with either

Discussion

Recent evidence from murine malaria and in vitro studies has suggested that platelets and platelet activation play a role in the pathogenesis of early malaria. Studies in humans are however sparse. From our current study, in which the decrease in platelet numbers was associated with an early increase in the IPF and TPO concentrations, we conclude that thrombopoiesis is not suppressed in early malaria, unlike the erythropoiesis. Increased peripheral platelet clearance thus seems responsible for the malaria-induced thrombocytopenia, a finding supported by an earlier scintigraphic platelet study showing a strongly reduced platelet life span in malaria patients [START_REF] Skudowitz | Mechanisms of thrombocytopenia in malignant tertian malaria[END_REF]. Second, we demonstrate that systemic platelet activation and consumptive coagulopathy are not responsible for this enhanced clearance.

Previously, in the same human malaria model, we have shown that endothelial cell activation with release of active VWF, i.e. VWF in a platelet binding conformation, is an early event in malaria and that VWF concentrations correlated negatively with platelet numbers [START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF]. Multiple other recent in vivo and in vitro studies have also implicated VWF release as a pathogenic mechanism in malaria [START_REF] Bridges | Rapid activation of endothelial cells enables P. falciparum adhesion to platelet decorated von Willebrand factor strings[END_REF]de Mast et al, 2009a;[START_REF] Larkin | Severe Plasmodium falciparum malaria is associated with circulating ultra-large von Willebrand multimers and ADAMTS13 inhibition[END_REF][START_REF] Lowenberg | Severe malaria is associated with a deficiency of von Willebrand factor cleaving protease, ADAMTS13[END_REF]. Given this presumed role for VWF, the increase in plasma concentrations of sGP1b is an interesting observation. sGP1b, also known as glycocalicin, is the external domain of the GP1b receptor, the main platelet receptor for VWF and the leukocyte integrin Mac-1 [START_REF] Wang | Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury[END_REF]. The external domain of the GP1b receptor can be shed from the platelet surface by cleavage by the metalloproteinase TNF-α-converting enzyme (TACE/ADAM17) and this leads to a clear attenuation of normal platelet function [START_REF] Bergmeier | Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo[END_REF]. Recently, Cheng and co-workers reported data from in vitro studies in which they showed that interaction of platelets with VWF under flow condition induces GPIb-ectodomain shedding, a process that could be inhibited by inhibitors of metalloproteases 15 [START_REF] Cheng | Shear-induced interaction of platelets with von Willebrand factor results in glycoprotein Ibalpha shedding[END_REF]. The similar kinetics of sGP1b and VWF plasma concentrations in experimental malaria and their positive correlation in Indonesian malaria patients support these recent findings by Cheng et al. This novel mechanism may not only explain the elevated sGP1b concentrations in absence of other signs of platelet activation in our volunteers, but may also prove important for the negative regulation of both excessive platelet adhesion and VWF-mediated cytoadherence of infected red blood cells -a mechanism that has recently been reported [START_REF] Bridges | Rapid activation of endothelial cells enables P. falciparum adhesion to platelet decorated von Willebrand factor strings[END_REF]. Future studies will need to establish the functional consequences of GP1b shedding in malaria.

There is a paucity of data on the occurrence of platelet activation in human malaria. To our knowledge, no previous studies have used flow cytometry to examine platelet activation in malaria patients. Our findings suggest that platelet activation does not contribute to the thrombocytopenia in early malaria. We cannot exclude that some degree of platelet activation occurs in severe malaria, a condition associated with organ dysfunction and high mortality.

No significant difference in absolute CXCL4 (PF4) and CXCL7 (β-TG) levels between patients with severe malaria and healthy controls was found in a well performed study in Thailand, in which special attention was paid to prevent ex vivo platelet activation [START_REF] Supanaranond | In-vivo platelet activation and anomalous thrombospondin levels in severe falciparum malaria[END_REF]. The occurrence of some low grade in vivo platelet activation was suggested, however, by the fact that malaria patients had a lower circulating platelet mass and a slightly higher CXCL4/CXCL7 ratio. Thus, while our study found no platelet activation in early malaria, further studies are required to clarify the role of platelet activation in more advanced stages of malaria. In contrast to studies in humans, marked platelet activation has been described in several malaria animal models (e.g. von Zur [START_REF] Von Zur Muhlen | Visualization of activated platelets by targeted magnetic resonance imaging utilizing conformation-specific antibodies against glycoprotein IIb/IIIa[END_REF]. There are however important differences in the pathology of (cerebral) murine malaria compared with severe human malaria. Murine malaria is primarily an immunopathological process with marked inflammation and little or no sequestration of pRBCs, while endothelial activation with pRBC sequestration is a central feature of human malaria [START_REF] White | The murine cerebral malaria phenomenon[END_REF]. These fundamental differences in pathology can explain the discrepancy in results of platelet activation studies between human and murine malaria.

These findings in human volunteers were supported by the finding of in vitro studies, in which incubation of whole blood with pRBCs failed to induce direct platelet activation or platelet sensitization to ADP. In contrast to our results, enhanced platelet aggregation and increased surface expression of CD62P and PAC-1 after incubation of platelets with pRBCs were found in previous studies [START_REF] Inyang | Platelet reactions after interaction with cultured Plasmodium falciparum infected erythrocytes[END_REF][START_REF] Srivastava | Platelet factor 4 mediates inflammation in experimental cerebral malaria[END_REF]. Differences in the used protocols may be responsible for these conflicting results. In vivo conditions were simulated as much as possible in our current study by using impedance aggregometry to measure aggregation of platelets after addition of RBCs, instead of PRP in optical aggregometry. In addition, parasite densities in the range of those normally found in clinical malaria infections were used, which limits the possible confounding effects of excessive release of free hemoglobin and ADP, both potent platelet activators, from pRBCs during the incubation time.

In recent years, platelets were shown to express various TLRs, although their precise functional importance remains elusive [START_REF] Aslam | Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in vivo[END_REF][START_REF] Cognasse | Evidence of Toll-like receptor molecules on human platelets[END_REF]. P. falciparum contains known TLR2/4/9 ligands [START_REF] Parroche | Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9[END_REF][START_REF] Gowda | TLR-mediated cell signaling by malaria GPIs[END_REF]. Incubation of whole blood with pRBCs did not change the surface expression of TLR2/4/9, while incubation of whole blood with specific TLR2/4/9 agonists did not upregulate CD62P expression. Two earlier studies also found no induced platelet CD62P expression after stimulation with TLR2 and TLR4 agonists [START_REF] Cognasse | Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets[END_REF][START_REF] Ward | Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor[END_REF]. Therefore, to date, it remains uncertain whether TLRs on platelets play a role in malaria pathogenesis. The contribution of activation of the coagulation cascade to the malaria-induced thrombocytopenia has also been disputed in the past [START_REF] Francischetti | Does activation of the blood coagulation cascade have a role in malaria pathogenesis?[END_REF]. In our study, coagulation activation did occur, as indicated by the elevated D-dimer concentrations in 9 of 11 volunteers. However, a consumptive DIC did not develop, given the absence of a clear prolongation in the APTT in all except one volunteer and the high-normal fibrinogen concentrations. Earlier field studies had already shown that frank DIC is only rarely encountered [START_REF] Vreeken | Haemostatic defect in non-immune patients with falciparum malaria: no evidence of diffuse intravascular coagulation[END_REF]. So, coagulation activation does occur in early malaria, as it occurs in almost every infectious disease, but its role in the marked thrombocytopenia in malaria seems only minor. The same holds true for splenic pooling of platelets; a scintigraphy platelet study in malaria patients was consistent with diffuse platelet sequestration, instead of splenic or hepatic pooling [START_REF] Karanikas | Platelet kinetics and scintigraphic imaging in thrombocytopenic malaria patients[END_REF], while asplenic persons also develop pronounced thrombocytopenia when infected with malaria [START_REF] Demar | Plasmodium falciparum malaria in splenectomized patients: two case reports in French Guiana and a literature review[END_REF]. Finally, immune-mediated mechanisms have also been implicated in the pathogenesis of malaria-associated thrombocytopenia. Antiplatelet IgG antibody concentrations were reported to correlate with platelet number [START_REF] Kelton | Immune-mediated thrombocytopenia of malaria[END_REF], although this finding could not be confirmed in another study [START_REF] Looareesuwan | Thrombocytopenia in malaria[END_REF]. Antiphospholipid antibodies also occur in malaria patients. While antiplatelet antibodies may play a role in later stages of a malaria infection, it seems unlikely that they are responsible for the early decrease in platelet number when parasite densities are still extremely low. Previously, we found no induction of anticardiolipin antibodies in our experimental human malaria model [START_REF] Damoiseaux | Experimental infection with Plasmodium falciparum does not result in the induction of anticardiolipin antibodies in healthy volunteers[END_REF].

In conclusion, there is growing evidence that platelets play a distinct role in the pathogenesis of malaria during the early phases of the blood infection. Thrombocytopenia occurs in absence of platelet activation or consumptive coagulopathy. We hypothesize that the increase

  . Platelet kinetics and coagulation parameters were studied in 11 volunteers in the 2007 and 2008 experimental infection.

  CA); CD41 ECD (P2 clone), CD40 PE (MAB-89 clone) and CD36 FITC clone; all from Beckman Coulter, Immunotech, Marseille, France); TLR2 PE (CD282, TL2.1 clone), TLR4 PECy7 (CD284, HTA125 clone; eBioscience, San Diego, CA) and TLR9 FITC (CD289, 5G5 clone; Hycult Biotechnology Uden, the Netherlands). Analyses were performed using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Miami Florida). Platelets were identified by FSC/CD41 positive staining. Isotype and fluorochrome matched controls as well as non-stained samples were used to adjust the detectors.Aggregometry. Platelet aggregation was measured in an impedance dual channel aggregometer (Chrono-log, Model 570, Havertown, USA) and analyzed by the AggroLink software package. 750 µl of platelet rich plasma (PRP) was pre-warmed for 5 min at 37˚C in a magnetically stirred polycarbonate cuvette. After calibration, pRBCs or uRBCs were added (final hematocrit of 15%) and aggregation was monitored on two leads with ADP (22 µM) and PBS as positive and negative control, respectively. Whole blood impedance aggregometry was chosen over optical aggregometry because the preparatory steps required for the latter technique (separation of RBCs from the PRP by extra centrifugation steps), resulted in unwanted platelet activation.

  Differences between groups were analyzed by Student's T-test. All analyses were performed with SPSS version 16.0.

  Fig 1C). Young platelets are larger and the changes in the IPF paralleled changes in the mean platelet volume (MPV; Fig 1D) and the platelet large cell ratio (P-LCR; Fig 1E). The relative thrombocytosis at week 3 was associated with a low IPF and MPV. A transient increase in plasma TPO concentrations occurred during the drop in platelet count (Fig 1F). Platelet clumps were not observed, neither by automated hemocytometry, nor by morphological examination (data not shown). Finally, in contrast to the IPF, the reticulocyte fraction decreased in the first days after the thick smear became positive and the subsequent start of antimalarial treatment, suggesting that erythrocyte and platelet production are independent processes in patients with malaria (Fig 1C). Absence of consumptive coagulopathy. Activation of the coagulation cascade was evaluated in the same volunteers by measurement of D-dimers, fibrinogen and the APTT. These parameters were measured once at day 1 after start of antimalarial treatment, i.e. at the day platelet numbers declined markedly. Concentrations of D-dimers were above the upper

  Fig 3E). sGP1b is the extracellular domain of glycoprotein-1bα, the main platelet receptor for VWF. Previously, we have demonstrated a similar time course for VWF concentrations[START_REF] De Mast | Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria[END_REF]. sGP1b concentrations depend on the circulating platelet mass and the relatively high sGP1b 3 are probably due to the relative thrombocytosis at that time point.The increase in sGP1b during parasitemia and treatment was not a direct effect of artemetherlumefantrine on platelets, because aparasitemic volunteers treated by a standard course of artemether-lumefantrine showed no increase in sGP1b concentrations(Fig 3F).

  sGP1b concentrations per platelet and VWF concentrations (Fig 4B) and VWF activation factors (Fig 4C).Ex vivo studiesFlow cytometry. The effect of pRBCs on platelet phenotype was studied by measurement of the surface expression of various platelet receptors upon incubation of whole blood with pRBCs containing synchronous, mature asexual P. falciparum stages (final parasitemia of whole blood ~0.25%), pRBCs with asynchronous P. falciparum stages (final parasitemia ~5%) or pRBC lysate. Neither synchronized, nor asynchronous pRBCs changed CD62P 1A of supporting information; only data for synchronous mature pRBCs shown). Lysate of pRBCs and uRBCs increased CD62P expression to the same degree, probably owing to the platelet activating effects of ADP and haem from erythrocytes.

  intact or lysed pRBCs (Fig 1B-F of supporting information). Finally, no change in CD62P or TLR expression was observed upon incubation of whole blood with the specific TLR ligands Pam3Cys (TLR2), lipopolysaccharide (TLR4) or CPG repeat ODN (TLR9; results not shown). Aggregometry. The effect of direct interaction of platelets with pRBCs on platelet aggregation was studied with impedance aggregometry. Incubation of pRBCs with non-synchronized asexual stages (hematocrit 15%; parasitemia 15%) with PRP (ratio 1:1) did not induce any detectable platelet aggregation (Fig 2A of supporting information). In addition, there was no difference in the degree of platelet aggregation to 22 µM ADP between PRP incubated with pRBCs or uRBCs (Fig 2B of supporting information).

  from VWF-mediated GP1b shedding; a process that may prevent excessive adhesion of platelets and parasitized erythrocytes.

Fig 1 .

 1 Figure legends

Fig 2 .

 2 Fig 2. Time course of the mean fluorescence intensity (X-mean) of the platelet activation

Fig 3 .

 3 Fig 3. Time course of plasma concentrations of (A-E) platelet α-granule and membrane

Fig 4 .

 4 Fig 4. (A) Scatter dot plot with line at mean of plasma concentrations of soluble glycoprotein-

  Fig 2

  as the mean fluorescence intensity) of the platelet receptors CD62P (P-selectin), CD36, CD40 and Toll like receptors-2/4/9 upon incubation of whole citrate blood with synchronized, mature parasitized red blood cells (parasitemia ~0.25%, black line) and uninfected red blood cells (grey line) and their lysate. Isotype fluorochrome matched platelets were used as a negative control (dotted line). 158x160mm (96 x 96 DPI)Impedance aggregometry using platelet rich plasma (PRP) incubated with asynchronous, asexual parasitized red blood cells (pRBCs; parasitemia 15%; red line) or uninfected red blood cells (uRBCs; blue line). (A) Incubation of PRP did not result in platelet aggregation. (B) No difference in the degree of platelet aggregation was observed after incubation of PRP with pRBC or uRBC and subsequent stimulation with 22 µM ADP. Changes in impedance were 9 and 8 Ω for the pRBC and uRBC group, respectively. 118x54mm (150 x 150 DPI)

British Journal of Haematology

Acknowledgements

The authors would like to thank C.C. Hermsen, A.J. Luty, G.J. van Gemert, M. van de Vegte-Bolmer and the malaria CDL team for preparation of infected mosquitoes and their role in performance of the experimental human malaria infections. Dr. P. Beckers, T.A. Arens, N.

Huibers-van

Drunen and W. Arts of the malaria diagnostic laboratory; J. Wiersma for clinical assistance. N. Jacobs for the flow cytometry experiments and E. Liesting and J. Kulk for the haemocytometric analyses. P.B. Asih for assistance with the field study in Indonesia. S.A.E. Sebastian for technical assistance. This work was supported by a junior researcher grant of the Radboud University Nijmegen Medical Centre (QdM). The intervention studies were funded by a European Union FP5 program grant (project nr. QLK2-CT-2001-01886) and by the Dioraphte Foundation (VSM Malaria, project nr. 06-03-08-00).


Henrique L Gomes
email: hgomes@ualg.pt

Paulo R F Rocha

Asal Kiazadeh

Dago M De Leeuw

Stefan C J Meskers

Anomalous temperature dependence of the current in a metal-oxide-polymer resistive switching diode

Keywords: resistive switching, non-volatile memory, bistability, electrical properties, traps PACS: 73.20.Hb, 73.40.Qv 73.61.Ng

Metal-oxide polymer diodes exhibit non-volatile resistive switching. The currentvoltage characteristics have been studied as function of temperature. The lowconductance state follows a thermally activated behavior. The high-conductance state shows a multistep-like behavior and below 300 K an enormous positive temperature coefficient. This anomalous behavior contradicts the widely held view that switching is due to filaments that are formed reversibly by the diffusion of metal atoms. Instead, these findings together with small-signal impedance measurements indicate that creation and annihilation of filaments is controlled by filling of shallow traps localized in the oxide or at the oxide/polymer interface.

Introduction

Resistive switching has been observed in metal-insulator metal (MIM) diodes for a wide variety of insulator such as oxides, nanoparticles, organic or inorganic semiconductors, and currently attracts attention for application in future non-volatile memories [1]. We have recently shown that functional memories can be obtained by inserting a thin oxide layer into a polymer diode [2]. A yield of switching diodes of almost unity has been reported. Switching is a generic property of metal oxides [3][4][5][6], the polymer only acts as a current limiting series resistance [7]. The memory is formed by applying a high bias pulse to the pristine diode. After this so-called forming process, attributable to soft breakdown of the oxide [2], the memory can be switched reversibly between a high conductance ON-state and a low conductance OFF-state. Thermal imaging has shown that the conduction in the ON-state is filamentary in nature [8]. At present, the nature and formation of these filaments is not clear. Several models for the switching have been proposed such as metal filaments that rupture locally by Joule heating, [9,10] or trap controlled tunneling between metal islands [11].

In order to distinguish between these mechanisms, we focus on the temperature dependence of the current for an oxide-polymer switching device. The anomalous positive temperature coefficient (PTC) that we observe below 300 K, shows that more filaments become active when the device is cooled down. This observation contradicts explanations based on atomic diffusion. We argue that filaments switch on and off by filling and emptying of shallow trap states located in the oxide layer or at the polymer/oxide interface. This hypothesis is supported by small signal impedance measurements.

Experimental

Pristine Metal-Insulator-Metal (MIM) diodes consisted of an Al bottom electrode, a sputtered layer of Al 2 O 3 (10 nm), a spirofluorene polymer (80 nm), and a Ba/Al (5 nm / 100 nm) top electrode (see figure 1). The diodes with an active area of 9 mm 2 and 1 mm 2 were encapsulated to exclude atmospheric contamination. The current-voltage (I-V) curves were obtained using a Keithley 487 picoammeter voltage source and capacitance-frequency and capacitance-voltage (C-V) curves were obtained using an Agilent 4192 impedance analyzer. Temperature dependent measurements were carried out in a liquid helium cryostat (Advanced Research Systems, ARS -HC2).

Positive bias voltage refers to the bottom electrode being poled positive with respect to the top electrode.

Results and discussion

The pristine devices were turned into programmable resistive switching memories by applying a 12 V voltage ramp as described previously [2,12]. After forming, the devices exhibit the usual bistable J-V characteristics and a negative differential resistance (NDR) in the ON state, see figure 2. At room temperature, the memory can be switched between OFF and ON-state by applying voltage pulses with amplitudes corresponding to the top and bottom of the NDR [13], here about 3-4 V and 6-8 V respectively.

A stable OFF-state is induced by applying high bias voltage for a long time, in the order of minutes. The time necessary to induce a switching to the ON-state is now much longer than the time necessary to record a full J-V curve. In agreement with literature [14], the electrical current in the programmed OFF-state follows a thermally activated behavior (see figure 3). The Arrhenius plot of the current measured at 1V is shown in the inset; yielding activation energy of 66 meV for the temperature range of 280-200 K. Below 200 K, the current is not thermally activated.

Interestingly, the J-V characteristics for the ON-state show a large increase in the magitude of the current upon lowering the temperature of the diode. This behaviour is illustrated in figure 4. The increase in current is more pronounced at higher bias voltage, in the voltage range below the sharp onset of the NDR. The lower the temperature the higher is the swicthing-off volatge. The physical mechanims behind this shfit in voltage is under study.

To further explore this unusual temperature depedence, the diode was programmed into the ON-state at room temperature and then cooled down while applying a continous bias voltage (2 V). The magnitude of the current more than doubles in a temperature range of 150 degrees, see figure 5. This corresponds to a positive temperature coefficient (PTC) of the electrical resistivity, ≈0.01 K -1 , an anomalously large value when compared with typical values for metals ( = 0.0039 K -1 for Cu). Furthermore, in contrast to the smooth decrease of the resistivity in metals, the resistivity of the diodes lowers in a step-like fashion upon cooling.

The step-like nature of the rise in current, strongly suggest that additional conducting filaments become active at low temperature. In order to check for selfheating effects during the experiments, we also carried out in other sample cooling runs where the applied bias was temporarily removed for intervals of times ranging from a few seconds to minutes, allowing for thermal equilibration. During these times the current across the sample becomes zero. In these runs, a similar increase in conduction was observed as shown in figure 6. A small section of the curve is magnified to shown that the rise in current is trough small discrete events (see inset). Furthermore, the PTC of the current is independent of heating speed and direction of temperature ramping.

Thus self-heating effects can be disregarded. Small PTC effects were reported by others [14][15][16] and attributed to metallic filaments.

The abnormally large PTC cannot be explained by a metallic type of conduction.

The large and stepwise increase in current can be rationalized by the temperature dependence of trap occupancy. Here we assume that shallow trap sites around the Fermi level could control the activity of filaments. At room temperature they are depopulated by thermal emission. However, upon cooling the diode, the shallow traps will be gradually filled and activate filaments.

In order to test the reported hypothesis that these trap sites are located in the oxide, [10] we performed small-signal impedance measurements. Figure 7 shows the temperature dependence of the capacitance (C) and loss (G/ ) where G is the conductance and is the angular frequency. The low-frequency capacitance remains constant. At low frequencies the loss is proportional to 1/ , which implies a constant DC conductance. As shown in figure 7, upon decreasing the temperature, the low frequency conductance increases. This is in agreement with the PTC observed for the DC conductance as shown in figure 5. The temperature dependence of the loss recorded with a signal test frequency of 300 Hz is shown in figure 8. Similar to the DC measurements, the loss shows a discontinuous, step-like increase upon cooling.

In previous work [7], we have shown that the frequency dependent response of the diode can be modeled as a double RC circuit represented in the inset of figure 7. At low frequency the response is determined by the properties of the oxide layer, while at higher frequencies the polymer layer dominates. This equivalent circuit was used to fit the loss curves recorded at different temperatures (shown in figure 9). Since the measured capacitance does not change significantly with temperature, changes are mostly resistive and only located in the oxide layer (represented as R OX ). The dependence of R OX with temperature as estimated from the equivalent circuit is shown in the inset of figure 9. A decrease in R OX from 160 k (at 300 K) to 6.6 k (at 124 K) is required to provide a good fit to the data. The increase of the loss at low frequencies indicates that the oxide conductance increases upon cooling. Therefore, the trap sites active in switching are located in the oxide or at the oxide polymer interface.

For these memory devices, the presence of shallow traps cannot be detected by smallsignal impedance measurements because the large DC current across the sample mask the contribution of traps to the low-frequency capacitance.

Concerning to the chemical nature of the trap sites, we mention the possibility of a water-related defect. This defect is known to contribute to temperature dependent changes in electrical properties at temperatures around 200 K, related to a phase transition [17][18][19]. The fact that both the dielectric loss and DC current do not change significantly near 200 K suggests that the water-related traps do not contribute to the PTC effect. Furthermore, the PTC effect is still observed for temperatures as low as 130 K which is well below 200 K, temperature where the water related defect becomes a trap for charge carriers. This strongly suggests that although water-related traps maybe be present, they are not responsible for the PTC effect reported here.

In order to get insight into the trap characteristics, thermally stimulated currents were also attempted. However, switching diodes shown relatively noisy currents, which hinder the observation of discernible structure in thermally stimulated currents.

Conclusions

We note that the commonly reported view that filaments are created by the diffusion of metallic species from the electrode into the bulk region cannot explain the abnormal positive temperature coefficient. Instead we argue that in the forming process conducting paths are preformed. Here diffusion of metal atoms can be involved, as indicated by a number of experiments [5,16,20]. However, this does not imply that every time the memory switches, a filament is formed or ruptured by diffusion of atoms.

Instead, our findings support that preformed paths exist and that these are turned on and off by filling and emptying of trap states, acting as micro-switches. Equivalent circuit modeling shows that these micro-switches are physically located in or near the oxide layer.

Figure captions

Fig. 1 .

 1 Fig. 1. (a) Schematic diagram showing the physical structure of the memory device, (b)

Fig. 2 .

 2 Fig. 2. J-V characteristics of the device in the OFF and in the ON-state.

Fig. 3 .

 3 Fig. 3. Typical J-V characteristics for the OFF-state. The inset shows the Arrhenius plot

Fig. 4 .

 4 Fig. 4. Temperature dependence of the J-V curve of a diode programmed into the ON-

Fig. 5 .

 5 Fig. 5. The temperature dependence of the current in the high-conductance ON-state

Fig. 6 .

 6 Fig.6. The temperature dependence of the current in the high-conductance ON-state

Fig. 7 .

 7 Fig. 7. Frequency dependence of the capacitance (C) and the loss (G/ ) in the ON state.

Fig. 8 .

 8 Fig. 8. Low-frequency loss (G/ ), of the diode as function of temperature.

Fig. 9 .

 9 Fig. 9. Experimental measured loss (points) and theoretical fits (continuous lines) using

Figure 1 (Figure 2 Figure 9 (

 129 Figure 1 (Fig_1.tif)

Acknowledgements

We gratefully acknowledge the financial support received from the Dutch Polymer Institute (DPI), project 703, from Fundação para Ciência e Tecnologia (FCT) through the research Unit, Center of Electronics Optoelectronics and Telecommunications (CEOT), REEQ/601/EEI/2005 and the POCI 2010, FEDER.


Elmar C Fuchs
email: elmar.fuchs@wetsus.nl

L F Luewton

Agostinho

Adam Wexler

R Martijn Wagterveld

Jan Tuinstra

Jakob Woisetschläger

The behavior of a floating water bridge under reduced gravity conditions

When high voltage is applied to pure water filled into two beakers close to each other, a connection forms spontaneously, giving the impression of a floating water bridge [1]-[8]. This phenomenon is of special interest, since it comprises a number of phenomena currently tackled in modern water science. In this work, the behavior of this phenomenon under reduced gravity conditions during a parabolic flight is presented by the means of high speed imaging with fringe projection. An analysis of the behavior is presented and compared to theoretical considerations. Supplementary video clips for the sequences shown in figs. 2, 4, 5&6 as well as 7&8 are available.

Introduction

In 1893 Sir William Armstrong placed a cotton thread between two wine glasses filled with chemically pure water. After applying a high voltage, a watery connection formed between the two glasses, and after some time, the cotton thread was pulled into one of the glasses, leaving, for a few seconds, a rope of water suspended between the lips of the two glasses [START_REF] Armstrong | Electrical phenomena The Newcastle Literary and Philosophical Society[END_REF]. As gimmick from early days of electricity this experiment was handed down through history until the present authors learned about it from W. Uhlig, ETH Zürich [START_REF] Uhlig | Personal communication, Laboratory of Inorganic Chemistry[END_REF]. Although easy to reproduce, this watery Confidential: not for distribution. Submitted to IOP Publishing for peer review 10 August 2010 connection between the two beakers, which is further referred to as 'floating water bridge' holds a number of interesting static and dynamic phenomena [START_REF] Fuchs | [END_REF]- [8].

At macroscopic scale several of these phenomena can be explained by modern electrohydrodynamics, analyzing the motion of fluids in electric fields (see, e.g., the Maxwell pressure tensor considerations by Widom et al. [9], or the text book on Electrohydodynamics by Castellanos [START_REF] Castellanos | Electrohydrodynamics[END_REF]).

On the molecular scale water can be described by quantum mechanics (e.g. [11], [12]). The gap at mesoscopic scale is bridged by a number of theories including quantum mechanical entanglement and coherent structures in water, theories which are currently discussed (e.g. [13]- [17] for water in general, and [18] specifically for the water bridge). Previous experiments [START_REF] Fuchs | [END_REF] suggested a possible change of the water micro structure inside the water bridge; first neutron scattering experiments [5] showed no difference in the microdensity of a D 2 O bridge compared to the bulk; recent 2D neutron scattering experiments [6] indicated a preferred molecular orientation within a floating heavy water bridge; detailed optical investigations [7] suggested the existence of a mesoscopic bubble network within the water bridge; and a Raman scattering study on vertical water bridges reported on a polarized water structure induced by the electric field [19]. A comprehensive review about water bridge research was published recently [20]. The properties of water at mesoscopic scales have drawn special attention due to their suggested importance to human physiology [START_REF] Pollack | Cells, gels and the engine of life[END_REF].

In this work, the first reduced gravity experiments with the floating water bridge are presented.

The data gathered with high speed imaging and fringe projection are discussed, the overall behavior is described and compared to theoretical considerations.

Experimental

The experiments were carried out onboard the PH-NLZ Fairchild Metro II Research Aircraft of the NLR (Nationaal Lucht-en Ruimtevaartlaboratorium -The National Aerospace Laboratory of the Netherlands), Hangar 3, Schiphol, on a specially designed set-up (see Fig. 1). All apparatus were mounted on an aluminum alloy plate provided by the NLR using struts and mountings certified for aviation and safe for accelerations up to 9G. The set-up contained a constant current regulated high voltage power supply (0-20kV) with a 250kΩ/500MΩ voltage divider. Instead of beakers two closed bottles with an additional opening in the sidewall were used. The bottles were mounted on a movable stage in order to manually create water bridges of different length. The water bridge was formed between the sidewall openings of the bottles. The bottles were filled with milli-Q water (conductivity < 1µS/cm) up to ~2mm below these openings. For all experiments, the initial temperature of the water was ~19°C, which was the onboard air temperature. Due to the short duration of the experiments (< 1 min), no significant temperature change was to be expected [7]. As electrode material, 2 x 2 cm² platinum plates were submerged into the water. One electrode was raised to high potential (+20kV), the other was grounded to the aircraft's general ground. A 42nF ceramic capacitor was connected parallel to the electrodes.

Background lighting was achieved with an Advanced Illumination BL18120 LED light with a horizontal grid for the fringe projection technique. For the first experiment, the onboard gravity was measured using a gravity meter (compactly shown as "G" component in Fig. 1) consisting of a 1kg weight on top of a pressure dependent resistor which was supplied by a 9V battery. This resistor was put in series with a 470kΩ resistor. A Photron SA-1 high speed camera was synchronized to a Contec AD12-16U(PCI)EV data acquisition board so that images, applied voltage (the voltage drop over the 250kΩ resistor) and gravity (the voltage drop over the 470kΩ resistor) could be recorded simultaneously. Camera and data acquisition board were controlled via an HP laptop computer using the PVF Photron software. The electricity for the whole set-up was provided by a DC/AC converter which was connected to the aircraft's internal power bus. The bridge's diameter and length were determined by comparison with the (known) dimension of the bottles (and their openings, respectively). Under normal gravity conditions, the diameter was ~2.5 mm with fluctuations of about 0.5 mm, comparable to previous experiments [START_REF] Fuchs | [END_REF][4][5][6][7]. Three different experiments were carried out:

1.) A bridge was created under normal gravity conditions, then a short (~3 sec) parabolic maneuver was aviated. For this experiment, high speed imaging, gravity and the applied voltage were recorded. This experiment ended with the return of gravity.

2.) A bridge was created under normal gravity conditions, then a longer (~12 sec) parabolic maneuver was aviated. For this experiment, high speed images were recorded.

3.) A parabolic maneuver was aviated without a bridge, and the high voltage was applied during reduced gravity conditions. For this experiment, high speed images and applied voltage were recorded. The data acquisition started during the zero-G phase which lasted ~3 seconds and ended after the return of gravity.

For all experiments, the frame rate was 1000 frames per second with an exposure time of 1/1000 sec. When the data acquisition board was used, one data set per frame was recorded. The right bottle was always grounded (aircraft common ground), and the left bottle raised to high potential.

Results

The behavior of the bridge during experiment one is depicted in Fig. 2; gravity and applied voltage for this experiment are shown in Fig. 3. Typically, in order to get real microgravity conditions (~10 -6 g) during parabolic flights, the experimental package is isolate from the aircraft thus allowing it to float freely. For the set-up described, this was not possible due to safety reasons. Thus, when attached to the aircraft, fluctuations of a few tens to hundreds of mg are usually observed, which agrees with the gravity meter record in Fig 3 . Moreover, the data shows a short hold of the gravity at 0.5 of its normal value at ~2.85s. Presumably, this is either due to the software problem [START_REF] Nicholls | Photron Ldt[END_REF], or the weight in the gravity meter became momentarily stuck due to horizontal acceleration forces -or it was simply a bumpy parabola. However, a malfunction cannot be entirely excluded. Thus an average function was overlaid in Fig. 3, and actually both curves are in rough agreement with the behavior of the water in the bottles: It rises from image a to j and starts to come down again in image k.

Since the gravity meter did not work properly during this experiment, and not reliably at all during the other experiments, the authors plan to replace it with an accelerometer in the future.

The brief deprivation of gravity established an oscillatory process in the bridge. Its diameter first increased (a -f) and then decreased (g -j). After the return of gravity, it increased again (k-o).

This oscillation can also be seen in the applied voltage (blue curve, Fig. 3): as the bridge's diameter increases, its resistance and therefore the applied voltage drops as the current is kept constant by the power supply to 0.5 mA.

The arrangement for experiment two was similar to experiment one. An 8.4 mm bridge (~3 mm diameter) was created under normal gravity, then a longer zero-G maneuver was aviated. This was done in order to test if the bridge length could be increased over the normal maximum length for the voltage applied (2cm at 20kV, see Refs. [START_REF] Fuchs | [END_REF] and [7]). However, before the experiment could be completed, at a distance of 13.8mm, the bridge disappeared. This was probably due to the oscillation process which started in this experiment as well, but since the zero G phase lasted longer, instead of increasing its diameter again, the bridge separated entirely. This effect is shown in Fig. 4. First the bridge diameter increases (b-e), and then decreases (f-g) until it ruptures (h). A detailed observation of the rupture process is given in Fig. 5. Bridge failure under zero G resembles what happens when the high voltage is switched off under normal gravity or when detergents are added [START_REF] Uhlig | Personal communication, Laboratory of Inorganic Chemistry[END_REF]. First the diameter is decreased to ~0.2 mm (a-e). After that, pearl-like sections are formed in the water string (f) until it bursts into a series of droplets (g). Whereas under normal gravity conditions these droplets fall down [START_REF] Uhlig | Personal communication, Laboratory of Inorganic Chemistry[END_REF], here they fly along the electric field lines in both directions (h), partly bouncing off, with the remnants of the water bridge still clinging to the bottle walls. This effect is shown in detail in Fig. 6. A logical difference from a breakdown under normal gravity is that the remaining droplets experience the electric force only, thus their movement is horizontal only. This is demonstrated in Fig. 6: The bridge breaks up into four distinct droplets as indicated in image a: one directly in front of the second gridline [START_REF] Armstrong | Electrical phenomena The Newcastle Literary and Philosophical Society[END_REF], one between the second and the third grid line [START_REF] Uhlig | Personal communication, Laboratory of Inorganic Chemistry[END_REF], one between the third and the fourth grid line [START_REF] Fuchs | [END_REF], and a larger one on the fourth grid line (4). This behavior is called "semi-coalescent bouncing" and has been recently reported in electrohydrodynamic atomization experiments with water. A detailed analysis of this effect is given in ref. [START_REF] Agostinho | [END_REF].

In a third experiment, the formation of the bridge under reduced gravity conditions was monitored. The bottles were arranged close together (distance 7.8 mm) such that under normal gravity a bridge would undoubtedly form with the application of high voltage. The outcome is shown in Fig. 7. If high voltage is applied under zero G, the water bridge does not form (a).

During the return of gravity (from b on), the formation of multiple aqueous connections can be observed. The exact moment of bridge formation (between Fig. 7f andg)) is analyzed in more detail in Fig. 8. In the right (grounded) beaker, bridge formation starts with water running down the bottle walls (Fig. 8 b-d) which is accompanied by a rising of the water in the left (high voltage) bottle (c-d) connected to the falling water in the right (grounded) bottle. The small bridge visible in (d) is destabilized by the fluid discharge caused by the falling water (e); and the subsequent formation of a much larger aqueous connection (f-l) resulting in multiple bridges (Fig. 7i) which collapse under their own weight while new watery connections are constantly formed above them (Fig. 7j-l).

Discussion

The deprivation of gravity seems to have a profound effect on the floating water bridge: It is severely destabilized and, if exposed to reduced gravity conditions long enough, destroyed.

Moreover, reduced gravity inhibits bridge formation which only begins once gravity returns.

The most obvious reason for that could be that in zero G, there is no contact between the electrodes and the water in the bottles. This possibility, however, is ruled out by observations and considerations: Most important, in experiment one the electrode in the right-hand bottle is visible (see arrow in Fig. 2a): it is almost touching the bottom of the bottle. Since this depth was the same for all experiments, there was always contact between the electrodes and the bottle water.

Secondly, due to the hydrophilic electrode surface and the Sumoto effect [START_REF] Daba | [END_REF], a separation of the water from the electrodes is not expected. Third, during the last experiment, the bridge formation started with both falling and bottom water (see e.g. Fig. 8d ande), meaning that all the water was in contact with the electrode since previous experiments have shown that the bridge is not formed due to the applied field alone but requires an electric current flow [7].

The destabilization of the bridge starts with an increase in diameter which is followed by a decrease which may either lead to heavy oscillations (experiment one) or to the destruction of the bridge (experiment two). The authors plan to repeat these experiments in order to find out if a critical maximum life time under micro-gravity can be determined. From the present experiment, this time is roughly 1.5 s. Idealizing the bridge as a resistor-capacitor element, this time would represent the relaxation time of the RC circuit. This is slightly longer than the time delay observed during start-up in previous experiments (0.5 -1 s) [7]. Initially, a watery connection or 'capillary bridge' forms between the two beakers when a high voltage is applied; this is well known from electrowetting and is essentially an electrostatic phenomenon [START_REF] Mugele | Electrowetting: from basics to application[END_REF]. Once established, current begins to flow through the thin watery connection causing a magnetic field along the capillary bridge to rise which focuses the electric field resulting in a thick and stable water bridge. This process occurs within the above mentioned time-scale.

These results raise serious questions about the role of gravity in this phenomenon, since it is not straightforward that gravity is essential for both its formation and its stability. Previous experiments have shown that the water flows in spirals through the outer layer of the bridge [5], [7]. From basic hydrodynamics it is straight forward that the driving force for this rotation can be gravity. It has been suggested that the rotation stabilizes the bridge [5], and it has been observed that the speed of rotation decreases with increasing bridge diameter [7]. Thus an increase in diameter, as it was observed in experiment 1 and 2, is probably accompanied by a decrease in rotational speed. However, since the diameter is decreasing again afterwards, an increase of the rotational speed would be expected. Therefore, the authors plan additional experiments with tracer particles visualizing the rotation of the outer layer.

The break-up of the bridge is comparable to what happens if detergents are added to a bridge or if the voltage is shut off [START_REF] Fuchs | [END_REF] with the difference that under micro-gravity none of the droplets falls down. Since break-up droplets move in different directions, one can safely assume that they carry different charges, which indicates that there are both positive and negative charges flowing simultaneously through the water bridge or charge separation takes places during break-up. In this experiment, stable water remnants at the bottle openings could be observed in response to the absence of normal gravity, illustrating another novel effect which was recently discovered by Agostinho et al. [START_REF] Agostinho | [END_REF]: One of the droplets looses mass upon contact with the beaker water and is then ejected into the opposite direction. This is called "semi-coalescent bouncing" and involves charge transfer between droplet and "cone" (or water remnant, in this case) as well as recharging of the droplet followed by electrostatic repulsion. The process is described in the framework of electrospraying, thus requiring a conductive connection between the water and the electrode, and can therefore be seen as additional proof that the connection to power was never severed during this experiment.

Next to the changes of the bridge itself, a big effect of all experiments is the motion of the bulk liquid in the bottles. In Fig. 2, the liquid in the beakers remains mostly near the openings, though the interface is tilting due to a residual acceleration into the page. In this case, the bridge, though destabilized in the process, remains connected. In Fig. 4, it appears that the liquid begins moving toward the bridge from both sides, but by frame g most of the liquid has migrated away from the openings, causing the bridge to break.

The question why the bridge would break in such a way deserves detailed analysis: Without gravity, intermolecular forces (water-container interaction forces and surface tension), which are normally mostly overruled by it, become more prominent: These forces are responsible to provide adhesion between water and the glass surface and cohesion between water molecules. This is why water forms spheres in zero gravity, and why it runs up the glass wall of the bottles in this experiment. However, these forces and gravity are defeated by the electric forces of the water bridge when the high voltage is applied. Thus, a priori, one would expect a much more intense formation process without gravity, since now the electric forces only have to overcome intermolecular forces, thus allowing them to act more effectively. In other words, the water which initially moves toward the bridge (Fig. 4 a-f) is expected to flow along the electric field lines, thereby increasing the bridge's diameter. The experiment shows the opposite: The water runs upwards the beaker walls, around the openings -and starts to form jets only when gravity returns, not before (see Fig. 7). Moreover, not only the falling water does so, but a jet of upwards crawling water is formed although most of the water is not near the openings during the whole time. This happens only after the term "upwards" is defined again, as can be seen from Fig. 8d-h in the left beaker. It should be noted that the water level in the beakers is the same throughout the observation period, so the most obvious explanation for the observation -the falling water increasing the level to make contact with the electrodes again and/or creating the upwards moving jets starting the process -must most probably be excluded here as well.

The formation and stability of a floating water bridge is dependent on the careful balance of forces at play within the entire system. The principal forces are gravity, inter-molecular forces and the applied electric field. The reduction of gravity will affect a subset of these forces most notably the wetting or adhesive force. The wetting force is stronger than gravitational acceleration in water as evidenced by capillary rise. Under reduced gravity adhesion increases its contribution to the behavior of the whole system. The water is no longer restrained by gravity and climbs up the walls of the bottles. This increase in surface area raises the amount of work done on the system by surface tension. The electric field is also performing work in order to maintain a bridge. Once the contribution from the surface energy work function exceeds that of the electric field work function the formation of a floating water bridge is thermodynamically prohibited. Thus, in the absence of gravity if the bridge is in operation it ruptures, if no bridge exists it cannot form. With the onset of gravitational acceleration the water is pulled from the glass surface by an increase in the weight of the water column. This permits the weaker electric field to once again act on the water, initiating electrospray phenomena which ultimately lead to the formation of the bridge. Additionally, the disruption of a floating water bridge by the introduction of surfactants indicates that bridge formation can only take place when the surface energy of the system is within certain limits.

While the above discussion of the effect uses a conventional approach it must be mentioned that the electric field induces a polarization and self-arranging of water molecules in the bridge as observed by neutron scattering experiments [5], [6].

Conclusions

Fig. 1 :

 1 Fig. 1: Sketch of the zero-G water bridge set-up. The circles at center are the bottles filled with water, the grey squares are movable platforms. Abbreviations: HSC…High speed camera; G…gravity meter; LED… LED backlight; 9V… 9V power supply for the backlight. The voltmeter symbols show where the signals for the data acquisition board were taken from.

Fig. 2 :

 2 Fig. 2: The floating water bridge under decreasing gravity (a), reduced gravity (b-i), and increasing gravity again (jo). The time between the images is 200 ms. (see also Fig. 3), the white arrow in (a) shows the position of the grounded electrode.

Fig. 3 :

 3 Fig. 3: Gravity and applied high voltage during experiment one. The letters signify the recording time of the images shown in Fig. 2 (highlighted by the vertical lines below each letter), the horizontal lines represent the voltages of the gravity meter for normal gravity (1g) and zero gravity (0g), respectively.

Fig. 4 :

 4 Fig. 4: A bridge is formed under normal gravity conditions (a), then gravity is decreased (b-h). The time between the images is 200 ms.

Fig. 5 :

 5 Fig. 5: Break-up of the floating water bridge under zero-G conditions, bottle distance 13.8 mm. The time interval between the images is 200 ms (a-d) and 100 ms (e-h), respectively.

Fig. 6 :

 6 Fig. 6: Droplets bouncing off the water remains on both bottle edges after the breakdown of the floating water bridge under zero-G conditions. The time interval between the images is 1 ms.

Fig. 7 :

 7 Fig. 7: Bridge formation under returning gravity. No bridge formation was observed in the absence of gravity (a), however the onset of gravitational acceleration (b-l) yielded multiple aqueous connections. The time between the images is 20 ms.

Fig. 8 :

 8 Fig. 8: Detailed analysis of bridge formation under increasing gravity. The time between the images is 10 ms (a-d) and 2 ms (e-l), respectively.

Acknowledgements 16

With great pleasure, the authors wish to thank Profs. Marie-Claire Bellissent-Funel (Laboratoire Léon Brillouin, Saclay), Eshel Ben-Jacob (Tel Aviv University), Cees Buisman (Wetsus -Centre of Excellence for Sustainable Water Technology), Karl Gatterer (Graz University of Technology), Emilio Del Giudice (Universitá di Milano), Franz Heitmeir (Graz University of Technology), Jan C.M. Marijnissen (Delft University of Technology), Laurence Noirez (Laboratoire Léon Brillouin, CEA-CNRS/IRAMIS, CEA-Saclay), Gerald H. Pollack (University of Washington), José Teixeira (Laboratoire Léon Brillouin, CEA-CNRS/IRAMIS, CEA/Saclay), Giuseppe Vitiello (Universitá degli studi di Salerno), as well as Ingo Leusbrock and Astrid H. Paulitsch (Wetsus, Centre of Excellence for Sustainable Water Technology) for the ongoing discussion on the water bridge phenomenon (in alphabetic order).

Water bridge experiments performed under micro gravity helped to identify and confirm relevant time scales in the formation and destruction of this phenomenon. Previous experiments showed that under the influence of a sufficiently high voltage a Taylor cone forms on both sides from which the water is electrosprayed. This electrospray provides an aqueous connection between the water in the two beakers and a current starts to run through this watery connection causing a significant mass flow, mainly from the anode to cathode beaker [START_REF] Fuchs | [END_REF], [4], [7]. Under stable conditions water flows in both directions [7]. What is new is that under micro gravity this flow is halted, approximately at the same time-scale needed for bridge formation, i.e. the time from the first water connection (capillary bridge) to the thick and stable bridge. This indicates that a careful balance between gravity, adhesion, cohesion, and the applied electric field is necessary for the formation of a stable water bridge. But since the force measured between the beakers in previous experiments [4], [7] is larger than the electric drag force for water with a conductivity < 1µS/cm, additional stability might be added to the bridge by molecular orientation [6] or vibrational coupling [18], [26]. The interplay of these forces could be changed in reduced gravity conditions, thus destabilizing the bridge.

Obviously, more low gravity experiments with the floating water bridge are called for. We aim to improve the set-up in order to better control the position of the bulk liquid, using e.g. hydrophobic coating in the bottles. In order to avoid excessive g-jitter, we furthermore plan to do future experiments in a much lower gravity environment, e.g. by constructing a safe free-floating set-up inside of the plane. With these experiments, we intend to verify the hypotheses suggested in this work.

Special acknowledgements

The authors would like to thank the Gerrit Oudakker of Wetsus for his indestructible optimistic efforts which paved the way for this experiment. Consequently, we would like to thank the NLR for making this experiment possible, especially the pilots Ger Nielen and Wim Bonnee for fantastically aviating the parabolic manoeuvres. In addition, we would like to thank Prof. Cees Buisman of Wetsus for financial support.