text:
Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

INTRODUCTION
Neutrophil granulocytes have a short lifespan. They undergo apoptosis within a few hours and are cleared from the circulation in the liver, spleen and bone marrow. 1 At sites of infection and inflammation, their lifespan is prolonged. 2 However, after having fulfilled their functions, large numbers of neutrophils undergo apoptosis at the site of infection/inflammation. Apoptotic cell death is generally characterized by chromatin condensation and fragmentation, cell shrinkage, blebbing of the plasma membrane, formation of apoptotic bodies, activation of caspase-3 and presentation of 'find-me' and 'eat-me' signals. A fundamental feature of apoptotic cell death is the maintenance of membrane integrity in order to prevent leaking of toxic cellular contents. 3 However, the integrity of the cell membrane of apoptotic neutrophils cannot be maintained for an extended period of time. Consequently, in the case of insufficient clearance, apoptotic neutrophils undergo secondary necrosis. Necrosis is defined by cell lysis, followed by the release of DAMPs (dangerassociated molecular pattern molecules), which results in the activation of inflammatory and immune processes. Whereas primary necrosis is induced by highly toxic substances, leading to the swelling and consequent lysis of cells, secondary necrosis is the consequence of apoptotic cells losing their membrane integrity. Therefore, major differences exist between the release of DAMPs from cells undergoing primary necrosis and that from cells undergoing secondary necrosis. In particular, secondary necrotic cells release considerably less ATP, although they release activated caspase-3 and proteolytically processed autoantigens. [4][5][6] As fast-acting effector cells of the innate immune system, neutrophils are rapidly recruited to sites of infection, where they exert their antimicrobial function. 7,8 To enable this rapid action, neutrophils harbor preformed antimicrobial effector molecules, such as defensins, lysozyme and cathelicidins, which can act immediately after cell activation without a need for timeconsuming de novo synthesis. 9,10 Therefore, many of the preformed substances are antimicrobial effector molecules. In addition, neutrophils also contain preformed cytokines, including CXCL8 11 and CXCL2, 12 which have important roles in the rapid recruitment of inflammatory cells to sites of injury or infection.
In the present study, we searched for additional preformed mediators of inflammation and identified interleukin (IL)-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in primary human neutrophils. Western blot analysis and confocal microscopy revealed that both IL-16 and MIF are stored in the cytosol rather than in neutrophil granules. We showed that IL-16 is processed in a caspase-3-dependent manner in apoptotic neutrophils, giving rise to the biologically active C-terminal fragment, IL-16C. Importantly, the release of both IL-16 and MIF correlates strongly with the secondary necrosis of neutrophils. We were not able to identify any stimuli that induced the release of IL-16 and MIF independent of neutrophil secondary necrosis. Therefore, IL-16 and MIF represent potential mediators and modulators of inflammatory and immune responses at sites of insufficient clearance of apoptotic neutrophils.

RESULTS
IL-16 and MIF are preformed cytokines in primary human neutrophils Mature neutrophils contain several preformed antimicrobial proteins. 13 In addition, some cytokines have been shown to be stored in mature neutrophils. 11,12,[14][15][16][17][18][19][20] To obtain broader insight into the preformed cytokines of human neutrophils, a lysate from freshly isolated primary human neutrophils was analyzed using the Proteome Profiler Human Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA). Out of the 36 cytokines, chemokines and acute-phase proteins screened, positive signals were obtained for the cytokines IL-1ra, IL-16, CXCL1 and MIF, indicating the intracellular presence of these cytokines in resting human neutrophils ( Figure 1). In addition, a signal for sICAM-1 was detected ( Figure 1). However, because a cell lysate rather than cell culture supernatant was analyzed with the Cytokine Array Kit in our study, the sICAM-1 signal likely indicates the presence of ICAM-1 in the neutrophil cell membrane rather than the soluble form of ICAM-1.
Preformed IL-16 and MIF are localized in the cytosol of human neutrophils Antimicrobial proteins represent the majority of preformed proteins in neutrophils. They are formed during granulopoiesis and stored in neutrophil granules. 21 Neutrophils possess at least four different types of granules: (i) azurophilic/primary granules, (ii) specific/secondary granules, (iii) gelatinase/tertiary granules and (iv) secretory vesicles. The various granule populations contain distinct sets of proteins, some of which are used as identification markers for the particular granule type.
IL-16 and MIF share a feature that is rather uncommon for cytokines: they both lack a signal sequence. 22,23 Having observed that these two cytokines with this unusual feature are present in neutrophils, we addressed the issue of the subcellular localization and release of IL-16 and MIF by primary human neutrophils in vitro by applying immunofluorescence staining and confocal fluorescence microscopy. Imaging confirmed the intracellular presence of preformed IL-16 and MIF in neutrophils ( Figure 2a). The white spots/areas shown represent the results of the computer-assisted analysis of colocalization of the green IL-16 signals with the red signals of S100A8 (Figure 2a, III and IV). The analysis showed a strong colocalization of IL-16 with S100A8, an abundant cytosolic protein in neutrophils (Figure 2a, IV). In contrast, colocalization analysis of IL-16 with gelatinase (MMP9; Figure 2a, I and II), a protein contained in both gelatinase and specific granules, showed a weak colocalization with IL-16. Similarly, a very strong colocalization between MIF and S100A8 signals was also observed ( The data thus indicate that IL-16 and MIF are stored in the cytosol rather than in the granules of neutrophils. To confirm this finding, subcellular fractions of neutrophils were analyzed using western blot analysis. The total cell homogenate as well as five subcellular fractions, that is, azurophilic granules, specific granules, gelatinase granules, secretory vesicles/plasma membrane and cytosol, were assessed for the presence of IL-16 and MIF. This analysis showed that IL-16 and MIF are present in the cytosol rather than in any of the granules or vesicles (Figure 2b). Therefore, this analysis clearly confirms the intracellular presence and cytosolic localization of both IL-16 and MIF in primary human neutrophils.
pre-IL-16 is processed in apoptotic neutrophils in a caspase-3-dependent manner IL-16 is synthesized as an~80-kDa precursor molecule, 24 recently termed pre-IL-16. 25 Previously, it was shown in CD4 + and CD8 + T cells that pre-IL-16 is cleaved enzymatically by caspase-3, 23,26 a cysteine-aspartic acid protease that has a major role in apoptosis. Caspase-3-mediated processing of pre-IL-16 results in the generation of the 17-kDa peptide IL-16C and the larger fragment pro-IL-16. 23 IL-16C is the secreted fragment, exerting chemotactic activity on CD4 + cells. 27,28 Neutrophils undergo spontaneous apoptosis within 40 h when cultured in vitro. 29 To investigate whether pre-IL-16 is cleaved into the biologically active IL-16C during neutrophil apoptosis, neutrophils were analyzed after 5, 10, 20, These results indicate that pre-IL-16 is indeed processed during apoptotic cell death in neutrophils.
Our results suggest that pre-IL-16 is cleaved by caspase-3, as has been observed in T cells. 26 To confirm this hypothesis, neutrophil apoptosis was induced by irradiation with 200 mJ/cm 2 UV light (256 nm). Immediately after irradiation, neutrophils were treated with a pan-caspase inhibitor (Q-VD-OPh), a caspase-3-specific inhibitor (Z-DEVD-FMK) and the solvent DMSO as control. After 6 h of incubation at 37°C, neutrophil viability was assessed with Annexin-V-FLUOS and PI staining. The intracellular levels of pre-IL-16 and IL-16C were assessed with western blot analysis.
UV light irradiation increased the apoptosis from~20 to~92%, inducing caspase-3 activation and pre-IL-16 processing into IL-16C after 6 h of incubation ( Figure 3c). Treatment with Q-VD-OPh and Z-DEVD-FMK significantly inhibited both apoptosis and the processing of pre-IL-16 into IL-16C by blocking the autocatalytic cleavage of the p20 fragment of caspase-3 into the p17 fragment ( Figure 3c). Processing and consequent activation of caspase-3 occur in a two-step process. 30 First, pro-caspase-3 is cleaved by the upstream caspases-8 and -9 into a heterotetramer complex consisting of two p20 and two p12 fragments. In the second step, the p20 fragments are autocatalytically processed into p17 fragments, forming the mature and biologically active p17/p12-caspase-3 complex. Our results show that it is the p17/p12-caspase-3 complex that mediates IL-16 processing in neutrophils. IL-16C and MIF are released from neutrophils during secondary necrosis Because IL-16 is processed during neutrophil apoptosis (Figure 3), we addressed the question of whether the IL-16C peptide was secreted by apoptotic neutrophils. Supernatants of in vitro cultured neutrophils were collected at various time points during 60 h of incubation, ultracentrifuged and their content of IL-16 and MIF determined. The proportion of apoptotic and necrotic cells at the indicated time points was assessed using flow cytometry following staining with Annexin-V-FLUOS and PI. In addition, the lactate dehydrogenase (LDH) enzyme activity was detected in the cell culture supernatants as a measure of cell death-associated release of cellular content. As early as 5 h of incubation,~10% of the cells were apoptotic; this proportion increased to~35% after 10 h and to more than 60% after 20 h of incubation (Figure 4a). Only a small proportion of necrotic cells was detected 20 h after incubation. After that, the proportion of necrotic cells increased gradually during the 60 h of culture ( Figure 4a). Similarly, LDH release was first detected after 20 h of culture. After this time point, the LDH levels of the culture supernatants increased with time, indicating enhanced cell death during prolonged in vitro culture (Figure 4a, top). Notably, IL-16 release did not have the same kinetics as apoptosis. Instead, IL-16 could be detected in the supernatants only after the appearance of secondary necrotic cells (Figure 4a). A very strong correlation was observed between IL-16 levels and the percentage of secondary necrotic cells as well as LDH release (Figures 4c and d, top). However, the correlation between IL-16 release and the proportion of apoptosis was notably weaker and not significant (Figure 4b, top).
Biologically active MIF does not require caspase-dependent proteolytic processing. However, in a previous study, MIF release was observed from apoptotic but not from viable neutrophils. 14 We assessed the release of MIF from neutrophils at various time points during the 60 h of in vitro culture. MIF release did not follow the kinetics of apoptosis but rather the kinetics of secondary necrosis of neutrophils (Figure 4a). MIF appeared in the supernatants only when necrotic cells and LDH release were detectable. Similar to IL-16, a very strong and significant correlation was  I and III), MIF (green, V and VII), MMP9 (red, I and V), S100A8 (red, III and VII) and the nuclei (DAPI, blue; I, III, V and VII). Staining was analyzed using confocal microscopy. Colocalization studies of the fluorescent signals for IL-16 and MMP9 (II), IL-16 and S100A8 (IV), MIF and MMP9 (VI), MIF and S100A8 (VIII) were performed. White signals indicate colocalization of the green and red fluorescent signals. Bars, 3 μm. The presented data are representative of three independent experiments. (b) Freshly isolated neutrophils were fractionated in a two-step Percoll gradient and analyzed for its content of IL-16, MIF, pro-MMP9 and S100A8 using western blot analysis. The presented data are representative of two experiments.
observed between the proportion of secondary necrotic cells and MIF release as well as between LDH release and MIF release (Figures 4c and d, bottom). Only a weak and nonsignificant correlation was observed between MIF release and the proportion of apoptosis (Figure 4b, bottom).
Our results revealed that IL-16C and MIF are released from secondary necrotic rather than apoptotic neutrophils. In an attempt to clarify the role of apoptosis in the release of IL-16C and MIF, we modulated neutrophil apoptosis. For this purpose, spontaneous neutrophil apoptosis was facilitated using UV-light irradiation (256 nm). UV-light irradiation enhanced neutrophil apoptosis in a dose-dependent manner (Figure 5a). Importantly, although irradiation with 200 mJ/cm 2 UV-light-induced apoptosis in~80% of neutrophils, no significant release of IL-16 and MIF was observed. However, irradiation with higher UV doses led to the release of high IL-16 and MIF levels ( Figure 5a). Notably, increasing the UV dose enhanced the degree of apoptosis only marginally but led to the induction of secondary necrosis in~25% of neutrophils ( Figure 5a). These results demonstrate once more that IL-16C and MIF are released not from apoptotic neutrophils but from secondary necrotic neutrophils, as has been shown during spontaneous cell death.
As another approach for modulating neutrophil apoptosis, neutrophils were infected with Anaplasma phagocytophilum, an intracellular bacterium that preferentially infects neutrophil granulocytes. Infection with A. phagocytophilum has been shown to inhibit neutrophil apoptosis. 31 As expected, after 6 h of incubation, neutrophils in the infected culture had a lower level of apoptosis (Figure 5b). Still, cells in the infected culture released significantly higher levels of IL-16 and apparently higher levels of MIF compared with uninfected neutrophils (Figure 5b). However, infected cells also contained a significantly higher proportion of necrotic neutrophils (Figure 5b), substantiating the view that the release of IL-16 and MIF is dependent on secondary necrosis rather than apoptosis.

DISCUSSION
Neutrophils are capable of expressing and secreting more than 50 different cytokines. 32 A few of these, such as IL-1ra, 20 IL-4, 17 IL-6, 15 CXCL8, 11 IL-12, 16 MIF, 14 CXCL2, 12 TGF-α 19 and VEGF, 33 are already produced during granulopoiesis and form pools of preformed cytokines in mature neutrophils. As the secretion of preformed cytokines is not delayed by prior de novo synthesis, these Using two gradient centrifugations and endotoxin-free reagents, we isolated neutrophils with minimal or no activation and contamination with other immune cells. In a lysate from these neutrophils, we identified the presence of CXCL1, IL-1ra, IL-16, MIF and sICAM-1 among the 36 cytokines and acute-phase proteins screened. This is in accordance with previous studies that also found IL-1ra 20 and MIF 14 to be preformed cytokines in human neutrophils. Moreover, CXCL2 (MIP-2), the murine homolog of CXCL1 and CXCL8, has been described to be a preformed cytokine in murine neutrophils. 12 The results of our array now confirm that CXCL1 is also preformed in human neutrophils. In contrast to this finding, our screening did not detect IL-6 and IL-12 among the preformed cytokines in human neutrophils, although the preformed presence of these cytokines has been described in murine neutrophils. 15,16 Furthermore, we could not confirm the intracellular storage of IL-4 17 and CXCL8, 11 as has been shown previously in human neutrophils. However, as these stores are supposed to contain only small amounts of IL-4 and CXCL8, the sensitivity of our array may not have been sufficient to detect these cytokines.
Although the presence of preformed IL-1ra 20 and MIF 14 has been described before in human neutrophils, IL-16 has not. There has even been doubt raised as to whether neutrophils express IL-16 at all. 32 In other cells, IL-16 is synthesized as a precursor molecule (pre-IL-16), which is cleaved by caspase-3 into the secreted and biologically active IL-16C. 23,24,26 If pre-IL-16 is a preformed cytokine in human neutrophils, it should also be processed during apoptosis. Indeed, our western blot analysis  showed that pre-IL-16 is cleaved into IL-16C during apoptosis of human neutrophils. Because caspase-3 inhibitors block pre-IL-16 cleavage in human neutrophils, the processing of pre-IL-16 is dependent on caspase-3 activation and presumably does not involve other proteases. Nonetheless, it has been shown in T cells that caspase-3 activation and pre-IL-16 processing are not automatically accompanied by apoptosis. We are not aware of apoptosis-independent activation of caspase-3 in neutrophils, nor were we able to detect a constant background caspase-3 activation, as postulated for CD8 + T cells. 26 Therefore, we assume that apoptosis, caspase-3 activation and pre-IL-16 processing are joint processes in human neutrophils.
Notably, in other studies, caspase-3 activation in the context of IL-16 processing was assessed by the appearance of the p20 fragment only. The p20 fragment of caspase-3 is a subunit of the p20:p12 heterodimer, which in a second step is autocatalytically cleaved into the p17 fragment, which forms the active p17:p12 caspase-3 heterodimer. 30 However, recent publications also indicate a biological function of the intermediate heterodimer p20:p12, which is able to activate PKCδ, which then mediates the NFκB-dependent synthesis of proinflammatory cytokines. 34 Our data suggest that pre-IL-16 in neutrophils is cleaved by the apoptosis-associated p17:p12 heterodimer rather than the inflammation-associated p20:p12 heterodimer of caspase-3.
Most of the preformed proteins of neutrophils are packaged into granules during granulopoiesis. However, this process is questionable for IL-16 and MIF. Both cytokines lack signal peptides and therefore do not enter the ER and the Golgi apparatus. Consequently, they do not follow the conventional secretory pathway. Examining the intracellular localization of IL-16 and MIF, we found both cytokines to be localized in the cytosol of neutrophils, along with the calcium-binding S100A8 protein. 35 This finding implies that IL-16 and MIF need to use unconventional pathways to be released, as described already for IL-1β 36 and FGF2. 37 Testing a panel of cytokines, microbial constituents and glucocorticoids in vitro, we were not able find a stimulus that induced an active release of IL-16 and MIF by neutrophils (data not shown). Our data suggest that IL-16C and MIF are released passively by secondary necrotic neutrophils rather than actively by apoptotic neutrophils. As necrostatin-1, an inhibitor of necroptosis, did not affect the ratio of apoptotic and necrotic cells during a 72-h in vitro culture (data not shown), necroptosis is unlikely to have a role in spontaneous cell death of neutrophils. Accordingly, necrostatin-1 did not inhibit IL-16 release of neutrophils (data not shown). The predication that IL-16C and MIF are released by secondary necrotic neutrophils is strengthened by our results that after UV light irradiation and infection with A. phagocytophilum, IL-16 and MIF release is in line with enhanced secondary necrosis, but not with apoptosis. This result contradicts the finding of Daryadel et al., 14 who showed that MIF is released by apoptotic neutrophils dependent on ABC transporters, indicating an active release. However, other studies also describe the active secretion of IL-16 and MIF after S100A8, 38 GM-CSF 38 and C5a 39 treatment. Repeating their protocols, we did not observe an elevated IL-16 and MIF release after treatment of our neutrophil preparations with S100A8, GM-CSF or C5a. On the contrary, as C5a and GM-CSF treatment decreased apoptosis and secondary necrosis after 24 h of incubation, we detected even less IL-16 and MIF in the supernatants of treated neutrophils (data not shown). Moreover, in studies by Riedemann et al. 40 and Daryadel et al. 14 the MIF concentrations in the supernatants of neutrophils are much higher than in our samples, reaching up to 8 ng/ml after 6 h of incubation of 5 × 10 6 cells. Because the MIF concentrations measured by us using similar settings were always less than 50 pg/ml, we believe that the high concentrations in the other studies may be because of another isolation protocol for neutrophils, leading to more contamination with other immune cells.
As glucocorticoids are known to be potent inducers of MIF secretion from monocytes, 41 we treated neutrophils with 1 μM-0.1 fM dexamethasone or hydrocortisone for 12 h. As described previously, we observed an anti-apoptotic effect 42 as well as a concomitant decrease in IL-16 and MIF release in the cells treated with higher concentrations of both glucocorticoids (data not shown). In our hands, treatment with commonly used pro-and antiinflammatory substances did not induce an active release of IL-16 and MIF by neutrophils; hence, the question arises of why these two cytokines exist as preformed cytokines when they are possibly only released during secondary necrosis. Although in the healthy organism apoptotic cells are immediately efferocytosed by professional phagocytes, this may not be the case during microbial and viral infections, as well as in autoimmune diseases. In this context, infection with HIV provides an interesting example. As H 2 O 2 produced by neutrophils is viricidal to HIV-1, 43 the virus impairs the function of H 2 O 2 release and accelerates the apoptosis of neutrophils. 44 Moreover, the HIV protein Nef inhibits the efferocytosis of apoptotic neutrophils by macrophages and monocytes. 45 Ineffective clearance of apoptotic neutrophils will cause the transition to secondary necrosis. As IL-16C was shown to inhibit the replication of HIV-1, 46,47 neutrophils may have found another way to reduce virus propagation by processing pre-IL-16 into IL-16C during apoptosis and releasing IL-16C during secondary necrosis. Moreover, impaired efferocytosis, 48,49 as well as elevated expression levels of IL-16 50,51 and MIF, 52,53 have been observed to be connected with increased disease severity in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Although MIF has been shown to have a critical role in recruiting inflammatory immune cells in diseases such as rheumatoid arthritis, 54,55 the biological role of IL-16 in autoimmune diseases still needs to be unraveled.
In conclusion, the present study demonstrates that IL-16 and MIF are preformed cytokines present in the cytosol of mature human neutrophils that are released exclusively during secondary necrosis ( Figure 6). Thus, it is plausible to assume that IL-16 and MIF contribute pivotally to the inflammatory immune reactions observed in diseases associated with defective clearance of senescent cells.

Isolation and culture of human neutrophils
Heparinized peripheral blood was obtained from healthy volunteers with written informed consent. All studies were approved by the ethics committee of the University of Lübeck (05-124). Granulocytes were isolated as described previously using discontinuous Percoll gradient centrifugation. 56 Granulocyte purity and viability were 499.9%, as assessed by differential staining with Diff-Quik (Medion Diagnostics, Düdingen, Switzerland) and Annexin-V-FLUOS/PI staining. Only preparations with o3% eosinophils were used for the experiments.

Preparation of neutrophilic subcellular fractions
Granule subsets of neutrophils were purified applying a three-layer Percoll density gradient, as specified by Kjeldsen et al. 57 Aliquots of each fraction were assayed for the presence of marker proteins by ELISA. 58 The fractions were pooled as follows: 1-7 were the α-band

Western blot analysis
For western blot analysis, neutrophils were lysed with 10% ice-cold trichloroacetic acid to prevent protein degradation by neutrophil proteases, as described previously. 59 Proteins were separated by reducing SDS-PAGE, transferred to a nitrocellulose membrane and blocked for 30 min at room temperature with TBST containing 5% BSA. The membranes were then probed with primary Abs (human/mouse/rat MIF Ab, 1 : 800; human IL-16 C-terminal peptide Ab, 1 : 1333; human S100A8 Ab, 1 : 800 or human/mouse caspase-3 Ab, 1 : 400; purchased from R&D Systems) at 4°C overnight. After washing, the membranes were probed with the corresponding secondary Abs (sheep IgG HRP-conjugated Ab, 1 : 1000; goat IgG HRP-conjugated Ab, 1 : 1000, R&D Systems) for 1 h at room temperature. After washing, the membranes were soaked with Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Signals of binding Abs were detected with the Fusion FX7 chemiluminescence reader and quantified using the Bio1D software (Vilber Lourmat, Eberhardzell, Germany). Equal loading of the gel was determined by reprobing the membranes with GAPDH (14C10) rabbit HRP-conjugated Ab, 1 : 1000 (Cell Signaling Technologies).

ELISA for IL-16 and MIF detection
Concentrations of human IL-16 and MIF in the supernatants were quantified using commercial ELISA kits (DuoSets, R&D Systems) according to the manufacturer's instructions. According to the manufacturer's information, the antibodies used in the human IL-16 DuoSet are directed against rhIL-16C (#AAC12732). Nevertheless, cross-reactivity with pre-IL-16 cannot be excluded, as the manufacturer did not test the detection of pre-IL-16. Assuming no resynthesis of pre-IL-16 during apoptosis, our own testing, using lysates (as prepared in the cytokine array) of 5 × 10 6 freshly isolated and MACS-enriched apoptotic neutrophils, indicates a 3.7 times stronger recognition of IL-16C than of pre-IL-16 (mean 710.9 pg/ml versus mean 192.6 pg/ml, n = 3, P = 0.040 as calculated by t-test for paired samples).

UV-light-induced apoptosis
To facilitate neutrophil apoptosis, 350 μl of 7×10 6 freshly isolated neutrophils were irradiated with 256 nm wavelength UV light (200-1600 mJ/cm 2 ) using a Stratalinker (Stratagene, Heidelberg, Germany). Subsequently, the irradiated cells were incubated at a concentration of 5 × 10 6 cells/ml in complete medium for 6 h at 37°C. IL-16C and MIF release from neutrophils S Roth et al Preparation of cell-free A. phagocytophilum and co-incubation with neutrophils The infection rate of A. phagocytophilum-exposed HL-60 cells was determined by counting the cells containing morulae in Diff-Quikstained cytospin preparations. When ≥ 40% of the cells were found to be infected, they were used for the preparation of cell-free A. phagocytophilum, as described previously. 60 Briefly, infected HL-60 cells were centrifuged at a speed of 250 × g for 10 min, resuspended in 2 ml of PBS and passed through a 25-gauge needle 10-14 times. Cellular debris was removed by pelleting at 750 × g for 10 min. The supernatant was collected and centrifuged at 2500 × g for 15 min. A. phagocytophilum obtained in this way was immediately resuspended in neutrophil suspension and incubated for 6 h at 37°C. For the infection of neutrophils, A. phagocytophilum derived from one infected HL-60 cell was used to infect one PMN. 31


extfieldsofstudy:
Materials Science
Medicine

text:
Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Introduction
The formation of a blood clot through the interaction of platelets, fibrin polymers, and the vessel wall is the primary mechanism by which the body arrests blood loss after injury. Platelet activation, fibrinogen polymerization and fibrin fiber crosslinking alter the mechanical properties of blood within minutes from those of a viscous fluid to that of a viscoelastic gel [1,2]. Effective blood clot formation therefore requires an increase in material stiffness, a characteristic that can be correlated with clinical coagulopathies [3,4].
Ex vivo blood clot mechanical properties have been correlated with a number of disease states. Inadequate clot stiffness, manifested as a 'soft' clot, is characteristic of hemorrhagic disorders such as hemophilia and has been correlated with surgical bleeding in cardiopulmonary bypass [5,6]. 'Hard' clots, by contrast, have been linked to the formation of thrombi in stroke, myocardial infarction, and deep vein thrombosis [1,[7][8][9][10].
The relationship between clot mechanics and vascular diseases underscores the importance of identifying the contributory roles of platelets and fibrin to hemostasis. Several ex vivo mechanical tests of clot stiffness have successfully transitioned into the clinic, most notably the thromboelastogram (TEG) and rotational thromboelastometry (ROTEM). Measurement of bulk clot stiffness by either the TEG or ROTEM can be structured as a differential test to assess the relative contributions of platelets and plasma factors to overall clot stiffness [11][12][13].
There are a number of new technologies that are being developed based on diverse approaches to look at the dynamics of blood clotting both in vivo and ex vivo. Dynamic light scattering analysis has been used to measure in vivo the clotting of blood by comparing motion of RBCs in flow or in stasis [14] and laser speckle rheology has been used to correlate changes in light scattering speckle intensity with viscoelastic property changes ex vivo [15]. A novel approach that combines laser detection of material motion and mechanical oscillation of the blood sample is RheoSpectris [16]. Ultrasound imaging has been used for detection of material strains in tissue following external application of force [17,18]. In addition there are multiple microelectromechanical systems (MEMS) technologies that incorporate microcantilever or piezoelectric approaches to measure the changes in material properties of blood ex vivo [19,20]. Recently developed high resolution measurements of individual platelet contractility and fibrin fiber stiffness have given additional insight into the cellular and molecular basis of clot mechanics, though single cell and molecule assessments have yet to be linked to global or bulk rheological properties of clots [9,21]. The majority of viscometric assessments of clot rheology require removal of a blood sample from the subject for analysis and therefore may not capture fully the pathophysiology of an arterial or venous clot in situ. Within this admittedly severe limitation, ex vivo approaches to studying blood clotting can give significant insight into detailed molecular and cellular mechanisms that regulate coagulation.
In contrast to approaches to assess ex vivo blood clot stiffness based on either light scattering or mechanical testing, ultrasound can assess tissue stiffness by generation of acoustic radiation force (ARF), which transfers momentum from the ultrasound waves to the propagating medium and any embedded reflective or absorptive objects [22,23]. ARF has been previously used to characterize the viscoelastic properties of various biological tissues including the liver, tumor tissue, and the vitreous body of the eye [24][25][26][27]. In a more recent development to assess viscoelastic properties of blood, ultrasound ARF is coupled with echo time delay estimation of red blood cell displacement in a technique called sonorheometry [28][29][30]. Other technologies that employ ultrasound in their approach to detect material properties of blood include supersonic shear imaging [31].
Here we describe the development of an optical ARF-based method of strain application to blood plasma clots such that the contributions of platelets and fibrin to its mechanical properties can be individually assessed (Fig 1a). Acoustically reflective microbeads were embedded in the clot to act as force transducers and strain gauges. Ultrasound ARF was then used to induce bead displacement within a plasma sample that was then tracked by video microscopy to evaluate clot stiffness (Fig 1b). The focal point of the ultrasound transducer corresponded to a microliter-sized region of a blood clot that was positioned away from container walls so that it acted on a relatively small numbers of platelet clusters within the 3D clot matrix. ARF was controlled to generate rapid and highly transient (subsecond) mechanical strains of a few 10s of microns to quantify local plasma clot stiffness. Because the method perturbed a small clot region and applied small strains, it measured blood clot stiffness at an intermediate length scale between the milliliter volumes of conventional rheometric approaches and single molecule and cell approaches such as optical tweezers or atomic force microscopy (AFM). ARF-based assessment of clot stiffness may therefore be useful for investigating the cooperative role of platelet signaling and fibrin dynamics in the elastic transformation of a blood clot.

Ethics statement
All participants provided written signed informed consent after explanation of study procedures. Experiments and study protocol as described in IRB-HSR # 12600 were approved by the Institutional Review Board for Health Sciences Research of the University of Virginia.

Experimental setup
The experimental setup (Fig 1a) consisted of a platform, which positioned the ultrasound transducer relative to the microscope objective at the transducer focal distance. The sample was held in place over the objective and acoustically coupled to the transducer with ultrasound gel (Aquasonic, Parker Laboratories, Fairfield, NJ, USA). The sample container was slightly larger than the transducer diameter in order to minimize beam interference with the container walls. A 2.54 cm square polystyrene box (8.2 mL) (Ted Pella, Redding CA, USA) was used to hold the fluid or plasma sample. An acoustically absorbent V-shaped polydimethylsiloxane form (see S1 Fig) was inserted into the box to minimize acoustic reflections and reduce the required sample volume to 2 mL. A 10 MHz single-element focused transducer (model IBMF103, NDT Systems, Huntington Beach, CA, USA) was coupled to the sample holder with ultrasound gel. The 10 MHz transducer had a diameter of 9.5 mm and a focal length of 19 mm. The transducer and sample holder were held in place by a custom stage mounted on an air table. The sample holder was mounted above a microscope (Diaphot 300, Nikon, Melville, NY, USA) with a 10 X microscope air objective, which permitted focusing into the sample at varying depths. For optimal foci alignment of the ultrasound beam with the objective plane of focus, the optical focal plane was fixed at 1300 μm above the sample holder surface for all stiffness measurements. The effect of different optical focal depths on bead displacement was investigated and the analysis is included in S2 Fig. The motion of beads dispersed within the sample was acquired at 30 fps by a video camera (Canon Vixia HF S21, Canon, Melville, NY, The ARF based measurement setup used a 10 MHz ultrasound transducer to apply ARF to blood plasma in which beads were suspended. A microscope with 10 X objective and video camera had a mutual focus with the ultrasound transducer so application of ARF to the sample was imaged. (b) To assess blood plasma stiffness: 1) Video microscopy of the blood plasma sample was collected during application of ARF, 2) The bead motion due to applied ARF was tracked by an ImageJ program, 3) Displacements during the ARF pulse interval were determined, and 4) The displacement data was analyzed for the sample's viscoelastic parameters.
USA) mounted to the microscope camera port. As beads experiencing ARF move more slowly than material shear waves due to their size relative to fundamental material components (e. g. molecules, fibers), standard frame rate (30 fps) video microscopy can be used to assess bead trajectories with a high degree of precision.

An acoustic beamforming model
The ultrasound beam intensity and pressure profiles were modeled using Field II (http://fieldii.dk/), a well characterized ultrasound simulation tool [32]. The example code provided by the designer for producing intensity and pressure profiles, calc_int.m, was modified for the specific transducer specifications and settings used in this study: transducer center frequency (10 Mz), transducer radius (9.525 mm), focal length (19.05 mm), cycles per pulse (8), pulse repetition frequency (7.5 kHz), number of elements (1). The transducer aperture was modeled using the function xdc_concave. The intensity was set to 202 W/m 2 in order to match the peak pressure 2.5MPa to that which was measured with the hydrophone. The program then generated simulated beam intensity and pressure profiles.
Bead tracking and processing of displacement data Two methods of single particle tracking implemented in the image processing software ImageJ (NIH, Bethesda, MD, USA) were used to quantify bead displacement due to incident ARF: a manual particle tracking protocol and an automated tracking algorithm [33]. Manual tracking was used when bead velocities were greater than 400 μm/s and the automated algorithm was used in all other cases. At high velocities manual tracking was more effective due to edge detection limitations in the automated tracking algorithm. The automated tracking algorithm utilized a cross-correlation technique to measure sub-pixel and sub-micron displacements [34].
Manual tracking was performed using the point tool in ImageJ with the "auto-measure" and "auto-next slice" features turned on. Determination of the elastic and viscous parameters of the Kelvin-Voigt viscoelastic model from position data was performed using curve fitting with the built-in function lsqcurvefit in the computational software Matlab (Natick, MA, USA) where the lower and upper bounds for viscosity (μ) and elasticity (k) were set to 0.1 and 10 pN·s·μm -1 and 0.1 and 10,000 pN·μm -1 , respectively.
Because beads like those used in optical ARF move much more slowly than material shear waves due to bead size relative to fundamental material components (e. g. molecules, fibers), standard frame rate (30 fps) video microscopy can be used to assess ARF induced bead motion.

Force calibration experiments
For determining stiffness and for modeling viscoelasticity, the magnitude of force applied must be known. The ARF was estimated by tracking beads and analyzing their trajectories and terminal velocities in known viscosity fluids. Stokes' law was applied to determine the force on an object moving through a fluid of known viscosity. We used 3 fluids with known viscosities, two NIST traceable viscosity standards (S3 and S6) (Cole Parmer, Court Vernon Hills, IL, USA) and blood plasma to calibrate ARF on the bead.
To estimate force, 15 μm diameter latex polystyrene beads were suspended in the fluid samples Polybead, Polysciences, Warrington, PA, USA). The S3 and S6 fluids have precisely known viscosities of 4.063 cP and 8.743 cP, respectively, at 25°C. At room temperature blood plasma has a viscosity of 1.6 ± 0.1 cP [35].
The sample holder was filled with a viscosity standard in which beads were suspended. The 10 MHz transducer used to generate ARF was triggered in all experiments (except where noted) by a custom amplifier circuit connected to a Macbook 2,1 laptop computer (Apple, Cupertino, CA) running a Matlab/C++ program that controlled the ultrasound transducer firing pattern. A 0.8 μs pulse length (8 wave cycles) was applied. We define the rate of pulse generation used to create an impulse sequence by a pulse repetition frequency (PRF). Pulses were generated at a PRF of 7.5 kHz over a pulse train interval of 0.5 s. Pulse train intervals were separated by 6 s of rest. In place of the custom amplifier circuit, a waveform generator and power amplifier were substituted to increase dynamic range in selected experiments.
To investigate the effect of changing the PRF on bead displacement the PRF was varied in the viscosity standards. PRFs of 15, 7.5, 3.75, and 1.875 kHz were used at a time interval of 0.5 s. The result was a total of 7500, 3750, 1875, and 937 pulses fired per burst for each respective condition.

Measurement and analysis of ultrasound attenuation in viscosity standards and in agarose gels
A 10 MHz ultrasound transducer for transmitting ultrasound signals and a signal receiver, either a bullet hydrophone (Onda Corporation, Sunnyvale, CA) or 10 MHz ultrasound transducer, were immersed in a degassed water bath. A two-axis micron precision stepper motor controlled the relative positions of the transmit transducer and the signal receiver (hydrophone or receive transducer). For measurement of ultrasound attenuation in the viscosity standards and in agarose, sample holders with varying acoustic path lengths were built (see S3 Fig). An arbitrary waveform generator AFG 3022B (Tektronix, Beaverton, OR, USA) and 55dB amplifier ENI A-150 (E & I, Rochester, NY, USA) was used to drive the transducer. A Lecroy 334A oscilloscope (Lecroy, Chestnut Ridge, NY, USA) collected waveforms from each transducer or hydrophone that were transferred by GPIB to computer for analysis. The attenuation was determined by comparing ultrasound signal amplitudes at different path lengths of viscosity standard and using analysis techniques described in a later section.
The total path length between the transmit and receive transducer or hydrophone was determined by the measurement of the time delay between the pulse transmission and the pulse receipt with the speed of sound in water (1497 m/s) [36]. Change in signal voltage due to signal path length within the material was determined by the average absolute value of the waveform. A trend of reduced signal voltage with material thickness was measured. The voltage attenuation data was used to form a system of equations specific for each thickness of material and solved for the attenuation coefficient of the material using the Matlab function lsqnonlin. The attenuation of water was 0.0022 dB·cm -1 ·MHz -1 [37].

Agarose gel formation
Agarose gel was made with UltraPure Agarose (Life Technologies, Grand Island, NY, USA). The agarose was added to Tris-Acetate-EDTA (TAE) buffer in proportions to generate a 0.5 wt % gel in an Erlenmeyer flask. The solution was heated for 30 s in the microwave and then poured into either a 2.54 cm or 10 cm square polystyrene box to a volume appropriate for the thickness of gel desired (8.2 mL or 500 mL, respectively). Agarose was allowed to cool for 1 h before removal from the mold and measurement of attenuation.

Agarose gel and blood plasma bead displacement measurement
The agarose gel was made as described above with the exception that 10 μL of 5 μm carboxylated YG fluorescent beads (2x10 6 )(Fluoresbrite, Polysciences, Warrington, PA, USA) were added. Similarly PRP was doped with 10 μL of 5 μm carboxylated YG fluorescent beads before initiating clotting. Both agarose and plasma gelled in molds for 1 h to maximize stability and permit transfer to a gel holder mounted in a water bath on the stage of the microscope. In this way acoustic attenuation was minimized to increase force transmittal and permit analysis of the much stiffer agarose gel. A ziplock bag was used to enclose each sample in its appropriate buffer (TAE in the case of agarose or Dulbecco's Phosphate Buffered Saline (DPBS) in the case of plasma). The sample was then aligned at the mutual focus of the 10 MHz transducer and a 40 X water immersion microscope objective. The fluorescent beads allowed imaging in a turbid gel. For generation of ARF the transducer was driven by an arbitrary waveform generator AFG 3022B (Tektronix, Beaverton, OR, USA) and 55dB amplifier ENI A-150 (E & I, Rochester, NY, USA).

Bead sedimentation velocity measurement
Bead sedimentation velocity was determined experimentally in citrated platelet rich plasma (PRP) by measuring bead time of flight over the thickness of a gasket in a flow chamber. The gasket thickness allowed for precise measurement of distance travelled due to gravity from one surface to the other.

Blood clotting experiments
Blood was collected from two healthy volunteers using the protocol described in IRB-HSR # 12600 in 2.7 mL citrated 3.2% Vacutainer tubes (BD, Franklin Lakes, NJ, USA). Blood drawn by venipuncture resulted in more variable levels of platelet activation, hence the use of Vacutainers for sample acquisition. The blood was centrifuged at 100 x G for 20 min to separate phases of PRP, buffy coat, and red blood cells. To obtain PRP, the supernatant of the centri-

Spatial distribution of acoustic radiation force
A focused ultrasound beam was aimed into a 2 mL sample volume container coupled acoustically via ultrasound gel (Fig 1a). In order to determine the focal point and shape of the beam, the pressure field was simulated in FIELD II (Fig 2a) [32]. Pressure was directly measured by hydrophone in a region spanning the analytically determined ultrasound focus (Fig 2b). A 2-D heat map defined the region of maximum pressure and the -6 dB beam boundaries (delineated by the dotted line). The -6 dB lateral beam width was 400 μm, which could be entirely observed within the 10 X microscope field of view (Fig 2b). The -6 dB axial beam length was 5 mm, resulting in a shallow pressure gradient relative to the optical field of view (2 mm in the axial direction), which allowed us to assume that the beads were in a relatively constant pressure field in the direction of wave propagation. Assumption of constant pressure field is supported by velocity data with viscosity standards, where terminal velocities (constant velocities for multiple tenths of a second during the pulse) are reached (Fig 3a).

Region of bead displacement
The bead displacements during application of a 7.5 kHz ARF impulse were tracked by video microscopy (Fig 1b). Bead displacements were measured by recording trajectories in a S6 viscosity standard (8.743 cP at room temperature). Maximum bead displacement (D m ) induced by an ARF impulse relative to position in the pressure field is shown in Fig 2c. Since the ultrasound beam has an approximately Gaussian power distribution with respect to its axis, we anticipated that there would be a gradient in the bead displacements across the microscope's 10 X objective field of view. As expected bead displacements were largest at the center of the transducer focus and smallest near the edges (Fig 2c). The average of the maximum bead displacement due to application of an ARF impulse (Dm avg ) was 206.2 +/-62.6 μm (n = 60). The distribution of D m across the field of view (1 x 2 mm) was approximately Gaussian (Fig 2d). Average bead displacement during the pulse approached a terminal velocity within 0.1 s of pulse initiation (Fig 2e).

Bead sedimentation
To control for bead sedimentation effects in clotting plasma prior to reaching the gel-point, we measured bead sedimentation velocity in citrated plasma. Bead sedimentation velocity was determined experimentally in anti-coagulated platelet rich plasma (PRP) by measuring settling time. The 15 μm polystyrene beads settled at an average velocity of 3.84 +/-0.15 μm/s (n = 250). The distance a bead fell during a 0.5 s pulse train was approximately 1.92 μm. The bead sedimentation distance constituted a 0.5% change in bead position within the -6 dB lateral beam depth (400 μm). Because bead sedimentation was small relative to the beam diameter, and sedimentation would be abolished once the clot was past the gel-point, it was assumed to negligibly influence observed bead trajectories. The microscope field of view was positioned on the beam focus by centering over the largest bead displacements. The ARF induced displacement of 15 μm beads was analyzed in the S6 viscosity standard (8.743 cP). Bead displacement was measured during the 0.6 s ARF pulse interval, Δt = t f -t i where maximum displacement D m was determined at t f .

Magnitude of force application
The magnitude of ARF applied to beads in clotting blood plasma was estimated by measuring displacements in citrated PRP and correlating motion to force using Stokes' law (Eq 1). Bead motion in citrate anticoagulated PRP was compared to that in viscous standards. Terminal bead velocities during ARF application were measured (Fig 3a) and Stokes' law for drag force was used to calculate the force applied to the beads (Fig 3b) [38]. Stokes' law is: where F is the force applied to the beads, μ is the fluid viscosity, R is the bead radius, and v is the bead terminal velocity [39]. The bead terminal velocities in the viscosity standards S6 (8.743 cP at room temperature) and S3 (4.063 cP at room temperature) and citrated PRP (1.6 cP at room temperature) [35] (Fig 3b). The differences in the applied force on the beads may reflect differences in the degree of ultrasound attenuation between the three fluids, so attenuation of the viscosity standards was measured.

The relationship between attenuation and force magnitude
All materials attenuate acoustic waves [22]. The attenuation in S6 and S3 viscosity standards and in the agarose elastic standard was measured to determine the influence of attenuation on force experienced by the beads. Acoustic signal amplitudes in the viscous and elastic standards were measured to determine attenuation and force applied to the beads for each material.
The attenuation coefficient of a material is described by the equation where V i is the voltage of the signal before entering the material, V f is the voltage of the signal after traveling a distance (l) through the material, f is the frequency of the signal, and α is the attenuation coefficient. The transducer frequency (f) was 10 MHz and the acoustic path length (l) in the sample was 1.9 cm. Change in signal voltage due to signal path length and material attenuation was determined by the average absolute value of the waveform. In all samples signal voltage decreased linearly with thickness. With the voltage attenuation data, a version of Eq 2 specific for each thickness of material was established. The system of equations was solved for the attenuation coefficient of the material using the Matlab function lsqnonlin. The attenuation of water was set to a previously reported value (0.0022 dB·cm -1 ·MHz -1 ) [37]. The attenuation coefficient of 0.5% agarose was found to be 0.0109 dB·cm -1 ·MHz -1 . The attenuation coefficients of the S3 and S6 viscosity standards were determined to be 0.0764 dB·cm -1 ·MHz -1 and 0.1056 dB·cm -1 ·MHz -1 , respectively. Eq 2, which describes attenuation of ultrasound signal voltage, can be extended to reflect a proportional change in applied force with the change in voltage across a signal path length within the material, such that where α is the material attenuation, F i is the ideal (non-attenuated) force delivered by the transducer, F f is the force calculated on beads in the material from Stokes' law (Eq 1) [40], V i is the voltage of the signal before entering the material, V f is the voltage of the signal after traveling a distance (l) through the material, and f is the transducer frequency. The lower ultrasound attenuation of S3 compared to S6 was consistent with the greater force experienced by beads in the S3 fluid than in the S6 fluid.
To determine the expected force differential between two materials a ratio of Eq 3 can be made. When the settings of applied ultrasound are the same for each material such that the F i in each material are equal where F material 1 is the force on beads in a material, F material 2 is the force on beads in a second material, α 1 and α 2 are the attenuation coefficients for the respective materials, f is the transducer frequency and l is the acoustic path length. The expected force differential between S6 and S3 viscosity standards was determined from the attenuation coefficients measured previously using Eq 4 such that The statistically bounded range of the F S6 to F S3 ratio using bead displacement analysis by Stokes' law was Since the ratio predicted by the fluid attenuations was within the Stokes analysis range, the agreement of the force and attenuation measurements suggested that fluid attenuation accounted for the difference in force (F f ) experienced by the beads in their respective fluids.
The lower force experienced by beads in citrated PRP relative to S3 or S6 standards predicts that blood plasma has a greater attenuation than S3 and S6 viscous standards, or even a 0.5% agarose gel. Previously reported attenuation coefficients (α) for unclotted and clotted blood plasma are 0.104 dB·cm -1 ·MHz -1 and 0.126 dB·cm -1 ·MHz -1 respectively [41]. The expected force differential between clotted and unclotted plasma was determined from the attenuation coefficients measured previously using Eq 4 such that As the relative difference between the estimated force acting on a bead in clotted and unclotted plasma differs by less than 10% based on the relationship between force and attenuation described in Eq 4, we chose to use the average of the two coefficients for all plasma conditions. Therefore, given the assumption of constant attenuation coefficient 0.115 dB·cm -1 ·MHz -1 , a constant force of 147 pN at 7.5 kHz PRF was assumed for all blood plasma material property measurements with the optical ARF technique.

The relationship between pulse repetition frequency (PRF) and force magnitude
Based on previous ARF theory [29], we hypothesized that varying the PRF (the number of ultrasound impulses applied within one second) would adjust the force applied to the beads and lead to a corresponding modulation of bead displacements. To test our hypothesis the PRF was varied in the S3 and S6 viscosity standards and in citrated PRP (Fig 3c). Material stiffness was correlated with PRF and bead displacement by the equation where D m was the maximum displacement of the bead after the ARF impulse, S was material stiffness, and PRF was the pulse repetition frequency of the material [28]. Reducing the PRF resulted in a linear reduction in the bead displacements, consistent with theory (Eq 8).

Viscoelastic measurement of platelet poor plasma (PPP) clotting
Fibrin contributions to stiffness were assessed by removing platelets from plasma through centrifugation to generate PPP. Bead displacement due to ARF pulsing decreased over the course of 17 min (Fig 4a-4c). Signs of increasing elasticity developed early after clot initiation; within 1 min of kaolin activation beads recoiled after removal of an ARF impulse (Fig 4a). Based on bead velocities, the PPP sample at 1 min had a Re~0.07 [39], suggesting that the sample was experiencing significant viscous damping of the developing elastic response, characteristics of a viscoelastic fluid. At later time points, the Reynolds number reduced further (Re < 0.002) as the viscosity increased and the velocity of the beads was reduced (Fig 4b and 4c). At 15 (Fig 4b) and 18 (Fig 4c) min the beads reached a plateau of maximum displacement during the ARF impulse, recoiling 100% after force application stopped, indicative of a high degree of material elasticity.

Viscoelastic model fitting of platelet poor plasma
Clot stiffness in PPP was determined by the equation: where S is the clot stiffness, F plasma is the ARF applied to beads in plasma, and D m is the average of bead maximum displacements after application of 0.5 s of an ARF pulse (Fig 5a). As a form of Hooke's law, the inverse relationship between applied force and resultant displacement defines stiffness (S) (Eq 9) [42]. Acoustic radiation force has been used in defining material stiffness in previous work [29]. The clot appeared to have near constant stiffness from 0-9 min and then stiffness increased 78-fold between 9 and 18 min. In order to quantify the changes in viscous and elastic components of the plasma during clotting the displacement data was fitted with the Kelvin-Voigt model. The Kelvin-Voigt model consists of a spring and a dashpot in parallel (Fig 5b). The spring constant, viscous dashpot constant, and the equation that defines the system together describe the displacement of the point of interest due to a given force. To evaluate the viscoelastic properties the characteristic equation for the Kelvin-Voigt model was fit to the displacement data during the application of a pulse train (see the Methods bead tracking section). The Kelvin-Voigt model captures the coupled effects of viscosity and material elasticity on force-induced material displacements and has been widely used in biological materials modeling [43].
Assumptions of the Kelvin-Voigt model include application of constant force, which is appropriate if the analysis is limited to the displacements measured during the pulse train interval. The force applied on the blood plasma sample can be considered constant during a pulse train interval because the ratio of the time constant of blood plasma viscoelastic response (~0.01 s) [44][45][46] to the pulse repetition period (~10 -4 s/cycle) is so large. Tissue (or blood clot) movement is negligible between each impulse, and therefore during the microsecond pulse train application the force is effectively constant on the tissue. Momentum of the bead, because of low Reynolds number, is assumed negligible and therefore not accounted for in the model.
The Kelvin-Voigt model was fit to bead displacement during force application to assess the viscoelastic behavior of blood [28,47,48]. Within a minute of PPP clot initiation spring-like behavior (displacement plateau during impulse) was evident and increased at all times measured (Fig 5c and 5d). In contrast, viscous fluid-like behavior (constant velocity during impulse) was observed with bead motion in the S6 viscosity standard at all time points measured. The elastic parameter k of S6 fluid, as expected, was near zero (S4 Fig). Viscous (μ) and elastic (k) parameters of PPP determined by the model at 24 min were 9.999 +/-0.001 pN·s·μm -1 and 2430 +/-1600 pN·μm -1 respectively (Fig 5e). This is an increase in viscous and elastic parameters of 6.9 fold and 1107 fold respectively from unclotted PPP. The clot properties changed most significantly between 6 and 15 min, when elasticity (k) rapidly increased.

Platelet depletion and clot stiffness
Stiffness of PPP samples was compared to that of PRP to assess platelet contributions to clot stiffness (Fig 5a). PRP increased clot stiffness 6.9 fold relative to PPP. Where significant elasticity developed, beads in both PPP and PRP recoiled after ARF induced displacement (Fig 4), a hallmark of reversible elastic deformation. Viscous (μ) and elastic (k) parameters determined by the model at 21 min in PRP are 6.364 +/-2.988 pN·s·μm -1 and 5127 +/-2440 pN·μm -1 respectively (Fig 5f). To assess the clot properties of PRP after initial hemostasis, the average fold difference of viscous (μ) and elastic (k) parameters of the clot as compared to PPP between 15 and 21 min were 1.3 fold and 11 fold larger, respectively. In PRP, elasticity and viscosity both developed most significantly between 9 and 15 min, paralleling the kinetics of viscosity and elasticity changes in clotting PPP. Video of PRP clotting is available in S1 Video. Measuring stiffness ratio of agarose elastic standard to blood plasma For interpretation of the measurements made by the optical ARF method relative to standard material property testing techniques, the stiffness of PRP was related to that of a 0.5 wt % agarose elastic standard. Both PRP and agarose have been measured by standard material property testing techniques such as the parallel plate rheometer. Stiffness comparison required the previously determined attenuation coefficients for plasma and agarose. The range of reported shear modulus of 0.5 wt% agarose is 1400-4530 Pa [49][50][51][52]. Previously reported shear moduli of PRP range from 406-600 Pa [11,53,54]. The ratio of the shear moduli of 0.5 wt% agarose to PRP as previously reported ranges from 2.3-11.2.
The ratio of stiffness of 0.5 wt% agarose and PRP measured by our optical ARF technique was compared directly to the ratio of previously reported shear moduli in order to validate the material property measurement capabilities of the optical ARF technique.
The expected force differential between PRP and agarose was determined from the attenuation coefficients measured previously using Eq 4 such that F plasma F agarose ¼ 10 À a plasma lf 10 10 À a agarose lf With the difference in force application between PRP and agarose determined, the displacements of beads in each material were measured. The beads in PRP were displaced 8.25 +/-0.91 μm and those in agarose were displaced 1.369 +/-0.13 μm. In order to compare the stiffness of the plasma to that of agarose the following equation was used where S agarose is the stiffness of agarose, S plasma is the stiffness of plasma, d agarose is the bead displacement in agarose, and d plasma is the bead displacement in plasma. Using Eq 11 the 0.5 wt % agarose gel was approximately 9.8 fold more stiff than a clotted PRP sample. This was within the ratio range (2.3-11.2) of the shear moduli reported previously for 0.5 wt% agarose and clotted PRP in independent determinations.

Measurement technique sensitivity to strength of clotting stimulus
Clotting of PRP was stimulated over a range of kaolin concentrations to assess the sensitivity of the optical ARF based clot stiffness assessment technique to strength of clotting stimulus (Fig 6). The measurements showed the capability of the ARF based bead-tracking technique to detect differences in kinetics of stiffness development that occur with variation in coagulation stimulus.

Discussion
In this study we describe an application of acoustic radiation force (ARF) to measure blood plasma clot stiffness. In this technique microbeads were embedded in the plasma to serve as coupled force transducers and strain gauges. ARF induced micron-scale displacements of acoustically reflective beads that were tracked by video microscopy. Bead forces were calibrated in viscosity standards to quantify viscoelasticity changes in blood plasma due to fibrin network formation. The motion of beads under ARF was initially dominated by blood plasma viscosity and later, as clotting progressed, was marked by increased elasticity. Comparison of the stiffness ratio of plasma and agarose gels measured by optical tracking of ARF-induced bead displacement to their previously reported shear moduli provided a point of reference to other rheometric techniques. Optical measurement of ARF-induced bead displacement (optical ARF) to assess blood plasma viscoelasticity was sensitive to biologically relevant characteristics of blood clotting such as strength of activation stimulus and platelet stiffness contributions. ARF generation requires ultrasound scatterers in the material to reflect and absorb energy. While the material itself experiences force, additional scatterers, such as embedded beads, significantly increase force application. ARF results from the millisecond integration of thousands of individual acoustic pressure waves into what is effectively a sustained impulse. Pressure wave integration occurs because the pulse repetition period (~10 -4 s/cycle) is much smaller than the time required for tissues to relax between acoustic pulses (~1 ms) [44][45][46]. Key to the utility of beads as force transducers are their greater attenuation of acoustic energy compared to the suspending medium, a difference of approximately 6-fold for polystyrene beads relative to plasma [55]. The bead radius is another significant determinant of optical ARF, as surface area is directly proportional to the resultant force [56]. A related application of ARF to blood clot stiffness estimation, sonorheometry, relies on the red blood cells at the beam focal point to reflect ultrasound and act as force transducers [28][29][30].
While material attenuation and bead radius influence the transmittal of ARF, ultrasound wave characteristics also affect the resultant force. Variation of pulse repetition frequency (PRF) was shown to proportionally modulate the force on the bead. By varying the PRF, the force per bead could be varied from 10-294 pN. These forces are on the order of those required for rupture of a fibrin fiber bond (200 pN) or to stretch a monomer in a fibrin fiber to 100% strain (140 pN) [57,58]. Since rupture of fibrin fibers in a network requires greater force than that needed to rupture a single fibrin bond, we assume the forces applied by ARF in this study to be largely non-destructive to the fibrin network, allowing evaluation of its viscoelasticity.
The assessment of plasma clot viscoelasticity by optical ARF was further validated by comparison with an elastic standard, agarose. Stiffness measurements of PRP, PPP and agarose were compared shear moduli determined in conventional rotational rheometers. In our case, agarose was determined by optical ARF to be 9.8-fold stiffer than PRP. This stiffness ratio was used to estimate a shear modulus ratio of agarose to PRP. Assuming the relationship was approximately proportional, the known shear modulus of 0.5% agarose ranging from 1400-4530 Pa would predict a shear modulus range of 143-462 Pa for PRP [49][50][51][52]59]. The estimate of the shear modulus for PRP was on the order of the 406-600 Pa range estimated in previous reports at equivalent times [11,53,54]. The 6.9 fold stiffness ratio of PRP to PPP measured by optical ARF was also of similar proportion to the reported stiffness ratio of 8.6 measured with the parallel plate rheometer [11]. Optical ARF estimates of stiffness therefore scales closely to a classic rheologic test and therefore can be extrapolated, within appropriate limitations, to a wider range of biologic testing of plasma blood clotting mechanics.
With the use of optical ARF there are several considerations that may confound the interpretation of the results. For instance, Brownian motion of the embedded bead could conceivably influence the estimation of the ARF-induced viscoelastic response. However, the diffusion coefficient of a 15 μm diameter bead in blood plasma was estimated to be 2.6 × 10-14 m 2 /s, and therefore the average bead would move only 0.33 μm/s [39,60,61]. Brownian motion was therefore considered to contribute negligibly in unclotted blood plasma relative to ARF induced bead displacement (>100 μm). Once a fibrin network has formed the bead diffusivity would drop even further. A related factor that could influence the interpretation of bead motion is the relative size of the beads compared to the fibrin network pore size and potential molecular interactions between bead and fibrin. However, polystyrene beads have been shown to non-specifically adhere to fibrin fibers [21] and the bead size (15 μm) is more than an order of magnitude larger than estimates of the fibrin mesh pore size (0.6 μm) [62], suggesting that the beads are likely trapped in the gel by adhesion and physical entanglement. Another potential concern is that bead motion under the influence of shear waves would be too fast to be captured at standard video rates. However, the maximum velocity at which beads moved when unconstrained in plasma was 600 μm/s, thus their frame to frame displacement was~20 μm-a distance easily resolvable by microscopy. A final point of consideration is the potential effect of the beads on the coagulation process. Embedding beads in a blood sample reduced time to clot and increased clot stiffness in a sonorheometry measurement (see S5 Fig), However, in our study, the presence of the beads was a constant and thus all samples experienced the same level of contact-pathway stimulation.
The detection of viscoelastic material properties by ARF-induced bead motion has several experimental limitations. For one, a microscope-based bead tracking approach has an inherent stiffness detection limit defined by video resolution and the optical signal to noise ratio; i.e, once the gel becomes very stiff relative to the force on the bead, motion stops. Further increases in gel stiffness are undetectable. However, improved signal detection can be achieved by increasing transducer power to increase bead displacement or by raising microscope magnification to increase spatial resolution. Increases in plasma clot turbidity, which becomes a significant optical limitation after 45 minutes, can also reduce bead displacement measurement accuracy. In this study, we used fluorescent beads to allow optical ARF analysis at advanced stages of PRP clot development (~1 h).
The mechanics of blood clots is complex. One of the more intriguing features of fibrin networks is their strain-hardening characteristic, or increased material stiffness with application of large strains (>10-100%) [58,[63][64][65]. A component of the strain hardening is thought to be a consequence of network deformation such that the slack is essentially taken out of the fibrin fibers. In platelet-rich plasma clots strain hardening does not occur [63], possibly by platelets pre-tensioning the network. Consequently, at low strains a PRP clot will have a larger elastic modulus than a PPP clot. But at progressively higher strains the difference between the two decreases as the PPP clot strain hardens, eventually becoming almost equivalent to PRP at strains of 20% and greater. Rotational rheometers such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM), which introduce strain at levels of 8-16%, could therefore potentially strain-harden of blood clots as they assess viscoelasticity. Under conditions where strain-hardening occurs, platelet contributions to clot stiffness may be less evident [53,66,67].
When PRP and PPP clots have been compared in the TEG assay, it has been observed that the stiffness ratio varies from 1.2-4 fold using TEG maximum amplitude (MA) assessment, depending on laboratory [68][69][70][71]. In contrast, the optical ARF method described here indicated the presence of platelets increased the clot stiffness by 6.9 fold, which was consistent with a previous study using ARF in whole blood [30]. The relative softness of the ARF probe described here, where induced strains were 2% or less, may therefore be useful in the analysis of platelet contributions to fibrin network stiffness, as sub-optimal platelet function would conceivably be more evident in the absence of fibrin network strain hardening.
Ex vivo measurements of clotting, such as the optical ARF method described in this study, almost all suffer a potential limitation in accuracy with regard to inferences of coagulopathies in vivo due to the fact that the blood sample is clotted under quiescent conditions. Since blood clot formation under stasis may increase fibrin and decrease platelet concentration as compared to in vivo arterial clots [72,73], it is important to qualify interpretations of clot mechanics depending on whether the test is based on quiescent or shear conditions, and additionally how the specific magnitude of shear might influence clotting; i.e., is the shear arterial or venous? With that consideration in mind, static condition measurement techniques, such as optical ARF, may be more appropriate when assessing coagulopathies characterized by venous flows, trauma, or surgical bleeding.
Optical ARF viscoelasticity measurements as described in this study appeared to be sensitive to both the contribution of platelets to clot stiffness and the level of clotting stimulus. Consequently, the approach could potentially be employed for studies on early stages of clot formation, which likely involves activation of the platelet contractile apparatus in addition to contributions of fibrin polymerization [74]. In addition, ARF measured platelet contributions to stiffness in a 3D clot matrix rather than on a surface, differentiating it from direct measures of platelet contractility such as micropost and AFM assays that typically require testing of isolated platelets on a substrate [75,76]. During clot formation, platelet contractile activity is translated to larger length scales through fibrin filaments and concurrent platelet aggregation. The ARF method we've described here may be useful in characterizing how single platelet force generation ultimately leads to the generation of tensioned fibrin networks and clot stiffness. The addition of beads to whole blood samples as measured by sonorheometry resulted in increased kinetics of clot stiffness and maximum clot stiffness as compared to whole blood samples without beads. (TIFF) S1 Video. Plasma clotting in optical acoustic radiation force clot stiffness assessment. Polystyrene microbeads (15 μm) in platelet rich plasma were displaced due to application of the acoustic radiation force. Bead displacement was reduced with increasing plasma clot viscoelasticity over the course of minutes. Clotting of platelet rich plasma was initiated with 5 μg/ mL kaolin.


extfieldsofstudy: Biology Medicine

text:
Inhibitory Effect of Duabanga grandiflora on MRSA Biofilm Formation via Prevention of Cell-Surface Attachment and PBP2a Production

Formation of biofilms is a major factor for nosocomial infections associated with methicillin-resistance Staphylococcus aureus (MRSA). This study was carried out to determine the ability of a fraction, F-10, derived from the plant Duabanga grandiflora to inhibit MRSA biofilm formation. Inhibition of biofilm production and microtiter attachment assays were employed to study the anti-biofilm activity of F-10, while latex agglutination test was performed to study the influence of F-10 on penicillin-binding protein 2a (PBP2a) level in MRSA biofilm. PBP2a is a protein that confers resistance to beta-lactam antibiotics. The results showed that, F-10 at minimum inhibitory concentration (MIC, 0.75 mg/mL) inhibited biofilm production by 66.10%; inhibited cell-surface attachment by more than 95%; and a reduced PBP2a level in the MRSA biofilm was observed. Although ampicilin was more effective in inhibiting biofilm production (MIC of 0.05 mg/mL, 84.49%) compared to F-10, the antibiotic was less effective in preventing cell-surface attachment. A higher level of PBP2a was detected in ampicillin-treated MRSA showing the development of further resistance in these colonies. This study has shown that F-10 possesses anti-biofilm activity, which can be attributed to its ability to reduce cell-surface attachment and attenuate the level of PBP2a that we postulated to play a crucial role in mediating biofilm formation.

Introduction
A number of reports have shown that bacterial cells growing in biofilms are profoundly resistant to many antibiotics. Biofilms play an intrinsic role in protecting bacterial cells from any fluctuations of the environment, including protecting the colonies from any potential antimicrobial agents [1]. It is well studied that the physiological properties of sessile biofilm populations are different from their planktonic counterparts and contribute to their better survival within the infected hosts. Biofilm-protected bacterial cells present a different mode of growth compared to planktonic cells, and the peculiarity of the mode of growth contributes to manifestation of antibiotic resistance. Due to this reason, treatment for biofilm-related infection becomes increasingly challenging, leading eventually to chronic device-related infections [2,3]. The biofilm forming ability of methicillin-resistance Staphylococcus aureus (MRSA) represents a major factor for nosocomial infections [1] and treatments for these infections are further complicated by the presence of other virulent factors such as toxin production and host immune evasion ability [4].
There are essentially two major steps in biofilm production: (1) cell-surface attachment, in which the bacteria attach to a surface in order to form colonies and this is also known as the primary attachment step; and (2) cell-cell interaction, which is an accumulative phase where the bacteria form microcolonies for construction of multilayer structure leading to biofilm development [5][6][7]. Biofilm formation in MRSA was previously reported to be mediated by PBP2a. PBP2a is an altered protein that evades antimicrobial action of beta-lactam antibiotics due to its low binding affinity [8]. It was hypothesized that PBP2a facilitates cell-cell interactions in the development of biofilm [9]. Hence, development of anti-biofilm agents that interfere with steps involved in biofilm formation and disrupt PBP2a expression would be a sensible approach in developing a new treatment for recalcitrant MRSA infections.
Duabanga grandiflora has been used traditionally to treat patients with skin conditions such as eczema or atopic dermatitis, who are also generally predisposed to Staphylococcus aureus infections [10,11]. In our earlier studies, we have reported that the ethyl acetate extract of the leaves of D. grandiflora possessed a broad spectrum antimicrobial action against a number of bacterial strains including MRSA [12,13]. The current study is aimed to investigate the effects of F-10, a bioactive fraction isolated from the leaves of D. grandiflora, on MRSA biofilm forming capacity and PBP2a expression in the biofilm.

Results
F-10 was tested at concentrations ranging from 0.75 mg/mL to 0.05 mg/mL and ampicillin at 0.05 mg/mL. The concentrations, 0.75 mg/mL and 0.05 mg/mL, were the minimum inhibitory concentrations (MICs) against MRSA growth in planktonic state for F-10 and ampicillin, respectively [14]. Figure 1 shows percentage of MRSA biofilm formation in the different treatments. Overall, F-10 exhibited appreciable activity against MRSA biofilm production, whereby biofilm production was at least 2-fold lower compared to control MRSA, while at the highest tested concentration of F-10 (0.75 mg/mL), the production of biofilm was down to 33.90%. On the other hand, ampicillin (0.05 mg/mL) reduced the biofilm production by more than 80% (Figure 1).

Figure 1.
MRSA biofilm formation (%) in microtiter plate wells containing F-10 (mg/mL) treatments. Three wells were used for each treatment. Experiment is representative of three independent tests, and error bars indicate the standard deviation. All difference between control and treated MRSA were statistically significant (p < 0.001).
Phytochemical analysis revealed the presence of alkaloids, tannins, saponins, steroids, glycosides and flavonoids in F-10. Preliminary HPLC analysis on the other hand confirmed that F-10 comprised a complex mixture of compounds ( Figure 2). In order to shed some light on the mechanism of anti-biofilm activity of F-10, cell-surface attachment was studied where MRSA cultures, treated either with ampicillin or F-10, were incubated for an hour. Cultures treated with F-10 showed a concentration dependent reduction in cell-surface attachment. Notably, at MIC of F-10 cell-surface attachment was markedly prevented (i.e., 5.33%), but the same cannot be said for that of ampicillin at MIC, which showed 62.20% of cell-surface attachment ( Figure 3).

Figure 3.
Attachment of MRSA cells to the surfaces of microtiter plate wells containing F-10 and ampicillin. Three wells were used for each treatment. Experiment is representative of three independent tests, and error bars indicate the standard deviation. All differences between control and treated MRSA were statistically significant (p < 0.001).
A PBP2a latex agglutination test was employed to measure semi-quantitatively the level of PBP2a found in the biofilm. A higher intensity of agglutination observed essentially corresponds to a higher level of PBP2a found in the biofilm. MRSA control showed a moderate intensity of agglutination while ampicillin treatment appeared to increase the amount of PBB2a in the biofilm. However, treatment with F-10, in all the concentrations used, resulted in a much lower agglutination of PBP2a (Table 1 and Figure 4). Table 1. Semi-quantitative estimation of PBP2a occurrence in biofilms isolated from different treatments. Intensity of agglutination against a turbid background was observed and scored between + to + + +, where the control latex which showed no reactivity in the absence of PBP2a is considered as "−" (interpretation: + + + high, + + moderate, + low, n = 3).

Discussion
Our study revealed that F-10, a semi-purified fraction isolated from D. grandiflora, possessed promising anti-MRSA activity via inhibition of biofilm production. From our preliminary phytochemical analysis of this fraction, the major classes of phytochemicals detected, which include alkaloids, tannins, saponins, steroids, glycosides and flavonoids, are known to be associated with antibacterial effects [15]. For instance, flavonoids are known to affect the sortase activity, which is known to influence the adhesive property of bacterial cell wall, leading to interruption of biofilm development [16,17]. Therefore, the occurrence of flavonoids in F-10 could represent a plausible explanation for the observed reduction of MRSA biofilm production when the cultures were treated with F-10. The anti-biofilm property of F-10 was further supported by the microtiter attachment assay results which showed that cell-surface attachment was effectively inhibited by F-10 (substantially better than ampicillin). This is a noteworthy finding, since the percentage of bacterial cell-surface attachment is directly proportional to the final mass of biofilm formed. A lower percentage of cell-surface attachment therefore corresponds to a lesser number of bacterial cells involved in biofilm development, which essentially gives rise to formation of weaker biofilm structures [18], and making the bacteria more vulnerable to eradication. Although ampicillin was found to suppress MRSA biofilm production, its inability to prevent cell-surface attachment within the first hour suggested a delayed antibacterial action. Previous studies have reported that a delayed antibacterial action may lead to prolonged and repeated exposure of the antibiotic to the bacteria, which promotes emergence of multidrug resistance [19,20]. Furthermore, the MIC of ampicillin used to treat the MRSA cultures was 50 μg/mL which exceeded the MIC breakpoints for susceptibility to beta-lactam drugs [21]. Therefore, ampicillin is not indicated for treatment of biofilm related infections.

Intensity of agglutination + + + + + +
In the PBP2a latex agglutination test, F-10 was observed to reduce the level of the resistant protein, PBP2a, in the MRSA biofilm. In a separate study, we have shown that F-10 attenuated the level of PBP2a in MRSA from a Western blot experiment [14]. It was previously suggested that the expression of high levels of PBP2a in MRSA could potentially promote cell-cell interactions that are not occurring between methicillin-sensitive Staphylococcus aureus (MSSA) cells, although the mechanism by which PBP2a promotes biofilm production remains unknown. It is well known that cell-cell interactions are vital for multilayer structure formation in development of biofilm [9,19]. We therefore postulated that the attenuation of PBP2a levels in MRSA by F-10, which will then negatively affect cell-cell interaction between MRSA cells that are involved in development of multilayer structures, consecutively contributed to the reduction in biofilm production.

Plant Material
D. grandiflora leaves were collected from a growing tree in Simpang Pulai, Pahang, Malaysia (GPS location: N04° 33.701' E101° 11.685'). Herbarium voucher specimens (herbarium code UNMC75) are deposited at the Herbarium of Faculty of Science, University of Nottingham Malaysia Campus.

Bacterial Strain, Growth Conditions and Determination of MIC Ampicillin
The bacterial strain used in this study was MRSA ATCC 43300. The strain was maintained on tryptic soy agar (TSA) (Hi-Media, Mumbai, India) supplemented with 2% NaCl (Merck, Darmstadt, Germany). All the experiments were initiated using fresh overnight cultures grown in tryptic soy broth (TSB) (Hi-Media) containing 1% glucose (Merck). MICs of ampicillin and F-10 against MRSA is 0.05 mg/mL and 0.75 mg/mL, respectively, which were determined based on methods described previously [14,23].

Inhibition of Biofilm Assay
Experiments were conducted based on methods described previously [20]. Basically, a 96-well microtiter plate was prepared with F-10 at the following concentrations; 0.75 mg/mL, 0.38 mg/mL, 0.19 mg/mL, 0.09 mg/mL, and 0.05 mg/mL, and ampicillin at 0.05 mg/mL. Aliquots of MRSA suspension were added to these wells. Final inoculum size was 1 × 10 5 CFU/mL in total volume of 200 μL in each well. The plate was incubated for 24 h at 35 °C. After incubation, the wells were washed with physiological buffered saline (PBS) solution and quantification of biofilm production was established by crystal violet staining method [24]. Experiment was done in triplicates on three separate occasions.

Microtiter Attachment Assay
Experiment were conducted based on methods previously described by Overhage et al. [18]. A 96-well microtiter plate was prepared with F-10 at the following concentrations; 0.75 mg/mL, 0.38 mg/mL, 0.19 mg/mL, 0.09 mg/mL, and 0.05 mg/mL, and ampicillin at 0.05 mg/mL. Aliquots of MRSA suspension were added to these wells. Final inoculum size was 1 × 10 7 CFU/mL. The plate was incubated for 1 h at 35 °C. Following incubation, the wells were washed with PBS and percentage of cell attachment was determined by crystal violet staining method [24]. Experiment was done in triplicates on three separate occasions.

PBP2a Latex Agglutination Test on MRSA Biofilm
MRSA was cultured in 50 mm diameter Petri dishes in 10 mL of TSB +1% glucose supplemented with F-10 at concentrations ranging from 0.05-0.75 mg/mL and ampicillin at 0.05 mg/mL. The petri dishes were incubated for 24 h at 35 °C. After incubation, the broth was carefully removed and 0.5 mL PBS was added to the petri dishes. Using a sterile 5 µL inoculating loop, the biofilm layer was scraped off just to fill the internal diameter (gives approximately 1.5 × 10 9 CFU/mL). The obtained bacterial biofilm was processed according to manufacturer's instructions of MRSA screening kit (Cat. No. DR900A Denka Seiken, Tokyo, Japan) in order to detect for the presence of PBP2a. Semi-quantitative estimation of PBP2a production in biofilms was done based on protocols described by Zhao et al. [25], in which the intensity of agglutination was observed and scored between + and + + +, where the control latex which showed no reactivity in the absence of PBP2a was scored as −.

Statistical Analysis
Results for inhibition of biofilm and microtiter attachment assays were shown as means ± standard deviation of three independent experiments. A one-way analysis of variance with Bonferroni multiple comparison tests were used to compare for difference between the control and treated groups. A p value of 0.001 was taken as statistically significant [20].

Conclusions
We conclude that F-10, the bioactive fraction obtained from D. grandiflora, inhibited biofilm formation in MRSA by preventing cell-surface attachment and interrupting PBP2a expression, which indirectly disrupts cell-cell interaction that is also necessary for biofilm development. Therefore it is postulated that PBP2a plays a crucial role in mediating the formation of biofilm which renders further resistance in these sessile biofilm populations of MRSA. Finally, some phytochemicals present in the bioactive fraction obtained from D. grandiflora, F-10, may be good candidates for development of new treatment for MRSA.