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Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo. The mechanisms underlying the activity of non-receptor tyrosine kinase, TNK1, in cancers are unclear. Here the authors show that MARK mediates 14-3-3 and TNK1 interaction which restrains TNK1 activity, while the release of TNK1 from 14-3-3 leads to TNK1 activation through its interaction with ubiquitin and thus results in TNK1-mediated tumor growth in vivo

A series of biphenyl per fluorocyclobutyl (BP-PFCB) polyethylene glycol (PEG) copolymers were prepared by nucleophilic addition the hydroxyl end groups of PEG to trifluorovinyl ether (TFVE) end groups of biphenyl perfluorocyclobutyl (BP-PFCB) oligomers. Upon addition, copolymers are linked through a new fluorinated arylene vinylene ether (FAVE) bond. Resulting copolymers were characterized by ATR-FTIR, 1H, and 19F NMR analysis. Gel permeation chromatography (GPC) showed a number-average molecular weights (M n s) of polymers from 9000 to 16,000. A moderated increase in M n s was observed compared to corresponding oligomers. Differential scanning calorimetry (DSC) thermograms revealed glass transition temperatures (T gs) from −25 to 127 °C. All copolymers showed one copolymer-specific T g. All T gs were compared to the theoretical values calculated from the Fox equation. Comparisons showed T gs became more agreeable at the lowest and highest PEG weight fractions. Thermal gravimetric analysis (TGA) studies showed a two-stage degradation in nitrogen and air that corresponded to copolymer weight percent. The onsets of degradations were from 343 to 360 °C for copolymers in air and from 333 to 343 °C for copolymers in nitrogen.

The development of an experimental protocol to investigate the flow field produced by the interaction of two immiscible liquids flowing through a porous network is reported. The experimental protocol allows simultaneous quantification of the velocity distribution in a multi-liquid system based on the microscopic particle image velocimetry technique. The experimental challenges associated with this unique application are discussed, including two-liquid imaging and interface tracking, and solutions that couple refractive index matching and fluorescent signal separation are described. The technique was applied to both single- and two-liquid flows in a two-dimensional pore network comprising a staggered array of circular pillars wherein the flow was driven by a steady pressure gradient. Both drainage and imbibition were considered herein with a focus on fluid–fluid front migration and effects owing to the passage of the interface. The velocity distribution obtained for these two-liquid-phase flow scenarios revealed several peculiarities when compared to the reference case of single-liquid-phase flow. In particular, the instabilities associated with the interfacial processes propagate downstream and perturb the flow field, resulting in dramatic differences from the regular and periodic flow paths typical of steady-state, single-phase flow. Additionally, the passage of the interface does not restore previous flow patterns, but instead yields complex preferential flow paths that mutually interact with residual trapped pockets of fluid. Such dynamical events must be quantified in order to properly model the pore-scale physics central to fully understanding the wealth of practical applications represented by this model flow system.

While conjugated polymer nanoparticles (CPNs) have been widely touted as ultra-bright labels for biological imaging, no direct comparative measurements of their intracellular brightness have been reported. Simple in vitro comparisons are not definitive since fluorophore brightness in vitro may not correspond with intracellular brightness. We have compared the fluorescence brightness of J774A.1 cells loaded with 24 nm methoxy-capped 2,000 M r polyethylene glycol lipid PFBT nanoparticles (PEG lipid-PFBT CPNs) to cells loaded with carboxy-functionalized quantum dots (Qdots) or a dextran-linked small molecule organic dye, Alexa Fluor 488 dextran (AF488-dex). Under conditions likely to be used for biological imaging or flow cytometry, these CPNs are 175× brighter than Qdots and 1,400× brighter than AF488-dex in cells. Evaluation of the minimum incubation concentration required for detection of nanoparticle fluorescence with a commercial flow cytometer indicated that the limit of detection for PEG lipid-PFBT CPNs was 19 pM (86 ppb), substantially lower than values obtained for Qdots (980 pM) or AF488-dex (11.2 nM). Investigation of the mechanism of cellular uptake of the three fluid-phase labels indicates that these particles are passively taken into macrophage cells via macropinocytosis without interaction with cell surface receptors, and ultimately localize in lysosomes. In addition, no cytotoxicity could be observed at any of the CPN concentrations tested. Together, these data suggest that these CPNs are appropriate and attractive candidates as fluid-phase markers with significantly greater fluorescence brightness than existing dyes or nanoparticles. We expect that these CPNs will find application in both imaging and flow cytometry.

This effort explores the impact of dilute polymer solutions on the turbulence characteristics in a submerged liquid impinging-jet configuration. Turbulent impinging jets are commonly used in technological applications such as drying, scouring, cooling, or heating due to an enhancement in transport characteristics in the impingement region under certain nozzle-to-wall configurations. Previous efforts have identified significant turbulence modifications in the presence of dilute concentrations of polymer in both bounded and unbounded flows, though the former has received considerably more attention. To this end, particle-image velocimetry measurements were taken for an axisymmetric turbulent impinging jet with a nozzle-to-wall distance H/D = 6.8 and nominal Reynolds number of 26,000. Measurements were performed for both plain water and dilute polymer solutions of polyethylene oxide at concentrations of 50 and 100 ppm. The mean and turbulence characteristics of these three flows are contrasted and it is observed that the two polymer solutions modify both the mean and turbulent characteristics of the jet in all three regions of interest (the free-jet, impingement, and wall-jet regions). Of interest, the 50 ppm case yielded a slight suppression of the turbulence in the free-jet region accompanied by a longer axial length of the potential core compared to the case of plain water. In contrast, the 100 ppm case exhibits clear enhancement of the turbulence in the free-jet region and a shortening of the potential core length. The effect of polymer was opposite in the impingement and wall-jet regions wherein the turbulence was slightly suppressed in the 100 ppm case in a manner consistent with the onset of the Toms effect in this wall-bounded region of the flow.

Barchan dunes are crescentic planform-shaped dunes that are present in many natural environments, and may occur either in isolation or in groups. This study uses high-resolution particle-image velocimetry (PIV) experiments using fixed-bed models to examine the effects of barchan dune interaction upon the flow field structure. The barchan dune models were created from an idealized contour map, the shape and dimensions of which were based upon previous empirical studies of dune morphology. The experimental setup comprised two, co-axially aligned, barchan dune models that were spaced at different distances apart. In this paper, two volumetric ratios (Vr, upstream dune: downstream dune) of 1.0 and 0.175 were examined. Models were placed in a boundary-layer wind tunnel and flow quantification was achieved via PIV measurements of the mean and turbulent flow field in the streamwise–wall-normal plane, along the centerline of the barchan(s), at an average flow Reynolds number of 59,000. The presence of an upstream barchan dune induces a “sheltering effect” on the flow. Flow on the stoss side of the downstream dune is controlled by the developing internal boundary layer from the upstream dune, as well as by the turbulent flow structures shed from the free shear layer of the upstream dune leeside. At both volumetric ratios, enhanced turbulence is present over the downstream barchan dune leeside, which is proposed to be caused by the interaction of shear layers from the upstream and downstream dunes. Both the size and magnitude of the shear layer formed in the leeside of the upstream dune control this interaction, together with the proximity of this shear layer to the stoss side of the downstream dune. Proper orthogonal decomposition (POD) analysis shows that the distribution of turbulent kinetic energy is shifted to higher modes (i.e., smaller spatial scales) over interacting barchan dunes, which also reflects the role of the leeside free shear layer in dominating the flow field by generation, or redistribution, of TKE to smaller scales.

The convective heat transfer behavior of laminar flow through a smooth- and two rough-wall microchannels is investigated by performing non-intrusive and spatially resolved measurements of fluid temperature via two-color fluorescent thermometry under constant heat flux conditions at three of the four microchannel walls. Pressure-drop measurements reveal that the apparent friction factors for all surfaces agree well with established macroscale predictions for laminar flow through rectangular ducts with the onset of transition at Re > Recr = 1,800 for smooth-wall flow and deviation from laminar behavior at progressively lower Re with increasing surface roughness. The local Nu for smooth-wall flow agrees well with macroscale predictions in both the thermally developing and developed regimes. With increasing roughness, while an enhancement in local Nu is noted for flow in the thermally developing regime, no measurable influence is noted upon attainment of a thermally developed state. These observations are supported by the examination of temperature profiles across the microchannel at various axial positions and Re, which suggest that the thermal boundary layer may be regenerated locally by roughness in the thermal entrance region of the flow resulting in an increased axial distance (compared to smooth-wall behavior) at which thermally developed flow is attained in the presence of roughness. Finally, estimates of the bulk Nu indicate enhancement in convective heat transfer over the smooth-wall case for laminar flow at higher Re while the smooth-wall bulk Nu data are found to agree well with macroscale predictions.

Modifications of fluid flow within microscale flow passages by internal surface roughness is investigated in the laminar, transitional, and turbulent regimes using pressure-drop measurements and instantaneous velocity fields acquired by microscopic particle-image velocimetry (micro-PIV). The microchannel under study is rectangular in cross-section with an aspect ratio of 1:2 (depth: width) and a hydraulic diameter of $$D_{\rm h} =600\,\upmu \hbox{m}.$$ Measurements are first performed under smooth-wall conditions to establish the baseline flow characteristics within the microchannel followed by measurements for two different rough-wall cases [with RMS roughness heights of $$7.51\,\upmu \hbox{m}$$ (0.0125D h) and $$15.1\,\upmu \hbox{m}$$ (0.025D h)]. The roughness patterns under consideration are unique in that they are reminiscent of surface irregularities one might encounter in practical microchannels due to imperfect fabrication methods. The pressure-drop results reveal the onset of transition above $$Re_{\rm cr}=1{,}800$$ for the smooth-wall case, consistent with the onset of transition at the macroscale, along with deviation from laminar behavior at progressively lower Re with increasing roughness. Mean velocity profiles computed from the micro-PIV ensembles at various Re for each surface condition confirm these trends, meaning $$Re_{\rm cr}$$ is a strong function of roughness. The ensembles of velocity fields at each Re and surface condition in the transitional regime are subdivided into fields embodying laminar behavior and fields containing disordered motions. This decomposition reveals a clear hastening of the flow toward a turbulent state due both to the roughness dependence of Re cr and an enhancement in the growth rate of the non-laminar fraction of the flow when the flow is in the early stages of transition. Nevertheless, the range of Re relative to Re cr over which the flow transitions from a laminar to a turbulent state is found to be essentially the same for all three surface conditions. From a structural viewpoint, instantaneous velocity fields embodying disordered behavior in the transitional regime are found to contain large-scale motions consistent with hairpin-vortex packets irrespective of surface condition. These observations are in accordance with the characteristics of transitional and turbulent flows at the macroscale and therefore indicate that the overall structural paradigm of the flow is relatively insensitive to roughness. From a quantitative viewpoint, however, the intensity of both the velocity fluctuations and structural activity appear to increase substantially with increasing roughness, particularly in the latter stages of transition. These differences are further supported by the trends of single-point statistics of the non-laminar ensembles and quadrant analysis in which an intensification of the velocity fluctuations by surface roughness is noted in the region close to the wall, particularly for the wall-normal fluctuations.

The character of transitional capillary flow is investigated using pressure-drop measurements and instantaneous velocity fields acquired by microscopic PIV in the streamwise–wall-normal plane of a 536 μm capillary over the Reynolds-number range 1,800 ≤ Re ≤ 3,400 in increments of 100. The pressure-drop measurements reveal a deviation from laminar behavior at Re = 1,900 with the differences between the measured and the predicted laminar-flow pressure drop increasing with increasing Re. These observations are consistent with the characteristics of the mean velocity profiles which begin to deviate from the parabolic laminar profile at Re = 1,900, interpreted as the onset of transition, by becoming increasingly flatter and fuller with increasing Re. A fully-turbulent state is attained at Re ≅ 3,400 where the mean velocity profile collapses onto the mean profile of fully-developed turbulent pipe flow from an existing direct numerical simulation at Re = 5,300. Examination of the instantaneous velocity fields acquired by micro-PIV in the range 1,900 ≤ Re

Microscopic particle-image velocimetry (micro-PIV) measurements are made in the streamwise–wall-normal plane of a 536 μm capillary at Re = 4,500 to study the statistical and structural features of wall turbulence at the microscale. Single-point velocity statistics, including the mean velocity profile, the root-mean-square streamwise and wall-normal velocities, and the Reynolds shear stress profile, agree well with established direct numerical simulations of turbulence in the same geometry at Re = 5,300. This consistency validates the efficacy of micro-PIV as an experimental tool for studying instantaneous, and even turbulent, flow behavior at the microscale. The instantaneous micro-PIV velocity fields reveal spanwise vortices that streamwise-align to form larger-scale interfaces that are inclined slightly away from the wall. These observations are entirely consistent with the signatures of hairpin vortices and hairpin vortex packets that are often noted in instantaneous PIV realizations of macroscale wall turbulence. Further, two-point velocity correlations and estimates of the conditionally averaged velocity field given the presence of a spanwise vortex indicate that hairpin structures and their organization into larger-scale vortex packets are statistically significant features of wall turbulence at the microscale.

High-resolution particle-image velocimetry (PIV) measurements are made in the streamwise - wall-normal plane of turbulent channel flow at Re$_{τ}$=566, 1184 and 1759, facilitating documentation of the population trends and core diameters of small-scale spanwise vortices. Swirling strength, an unambiguous vortex-identification criterion and hence a local marker of rotation, is used to extract small-scale spanwise vortex cores from the instantaneous velocity fields. Once the small-scale vortices are properly extracted from the PIV realizations, their characteristics are studied in detail. The present results indicate that the very-near-wall region (y < 0.1 h) is densely populated by spanwise vortices with clockwise (negative) rotation. This behavior supports the notion that hairpin-like vortices are generated very close to the wall and grow into the outer layer as they advect downstream. In contrast, counterclockwise (positive) spanwise vortices are scarce in the very-near-wall region, but their presence steadily increases within the logarithmic layer presumably due to a localized generation mechanism. The average core diameter of negative spanwise vortices is found to be larger than the average diameter of positive vortices, with few positive vortices having core diameters exceeding 80 y$_*$.

The influence of peak-locking errors on turbulence statistics computed from ensembles of PIV data is considered. PIV measurements are made in the streamwise–wall-normal plane of turbulent channel flow. The PIV images are interrogated in three distinct ways, generating ensembles of velocity fields with absolute, moderate, and minimal peak locking. Turbulence statistics computed for all three ensembles of data indicate a general sensitivity to peak locking in the single-point statistics, except for the mean velocity profile. Peak-locking errors propagate into the fluctuations of velocity, rendering single-point statistics inaccurate when severe peak locking is present. Multi-point correlations of both streamwise and wall-normal velocity are also found to be influenced by severe levels of peak locking. The displacement range of the measurement, defined by the PIV time delay, appears to affect the influence of peak-locking errors on turbulence statistics. Smaller displacement ranges, particularly those that produce displacement fluctuations that are less than one pixel in magnitude, yield inaccurate turbulence statistics in the presence of peak locking.

Acceleration is a fundamental quantity in fluid mechanics because it reflects the sum of all forces (pressure and viscous) present within the flow. However, measurements of acceleration have been difficult to achieve relative to the ease with which fluid velocity can be measured. A particle image accelerometer (PIA) has been developed to measure Eulerian acceleration fields by time-differencing successive measurements of the Eulerian velocity field as measured by particle image velocimetry (PIV). The measurements can also be made in uniformly translating frames. With current video camera technology, it is often not possible to measure the two velocity fields with a time separation sufficiently small enough to permit accurate finite difference approximation of the time derivative. A two-CCD-camera system has been developed to alleviate this limitation. Polarization filtering is utilized to separate the particle images viewed by each camera. The polarization filtering is achieved using cross-polarized light-sheets and a polarization filter just upstream of the imaging optics of the cameras. In this manner, PIV measurements can be achieved easily at time delays several orders of magnitude smaller than the shutter-time of the CCD cameras. The accuracy of the acceleration measurements is determined by numerical finite differencing errors and random noise and bias errors associated with the measurement of velocity. These errors, and methods of compensating for them, are studied.

We describe a new super-resolution method of particle-image velocimetry (PIV) based on Kalman filtering and χ2-testing. Performance of the “super-resolution KC” method is evaluated by Monte-Carlo simulation and by applying the method to measurements of flow fields recorded in the form of double-pulse/single-frame and single-pulse/double-frame particle images. When the images have good contrast, and depending on the intensity of the velocity gradients present in the flow, the super-resolution KC method is able to extract valid measurements from 80 to 100% of the available image pairs. In these tests, the vector yield is increased by more than five times compared to standard PIV analysis.

Methods of analyzing and interpreting velocity-field data (both two- and three-dimensional) to understand the kinematics, dynamics, and scales of turbulence are discussed. Reynolds decomposition and vorticity are traditionally used; however, several other methods, including Galilean (constant convection velocity) and LES decompositions (low-pass filtering), in conjunction with critical-point analysis of the local velocity gradient tensor, reveal more about the structure of turbulence. Once the small-scale structures have been identified, it is necessary to assess their importance to the overall dynamics of the turbulence by visualizing the motions they induce and the stresses they impose both on other small-scale vortices and on the larger-scale field.

Aggregation of suspended yeast cells in a small-scale ultrasonic standing wave field has been monitored and quantified. The aggregation effect is based on the acoustic radiation force, which concentrates the cells in clumps. The ultrasonic chamber employed (1.9 MHz, one wavelength pathlength) had a sonication volume of 60 microl. The aggregation process was observed from above the transducer through a transparent glass reflector. A distinct, reproducible, pattern of clumps formed rapidly in the sound field. The sound pressure was estimated experimentally to be of the order of 1 MPa. Microscopic observations of the formation of a single clump were recorded onto a PC. The time dependent movement patterns and travelling velocities of the cells during the aggregation process were extracted by particle image velocimetry analysis. A time dependent change was seen in the particle motion pattern during approach to its completion of clump formation after 45 s. Streaming eddies were set-up during the first couple of seconds. The scale of the eddies was consistent with Rayleigh micro-streaming theory. An increase in the travelling velocity of the cells was observed after 30 s from initially about 400 microm s(-1) to about 1 mm s(-1). The influence of a number of mechanisms on particle behaviour (e.g. micro-streaming, particle interactions and convective flow) is considered. The experimental set-up introduced here is a powerful tool for aggregation studies in ultrasonic standing waves and lays the foundation for future quantitative experiments on the individual contributions of the different mechanisms.

Exploratory measurements of oscillatory boundary layers were conducted over a smooth and two different rough beds spanning the laminar, transitional and turbulent flow regimes using a multi-camera 2D-PIV system in a small oscillatory-flow tunnel (Admiraal et al. in J Hydraul Res 44(4):437–450, 2006). Results show how the phase lag between bed shear stress and free-stream velocity is better defined when the integral of the momentum equation is used to estimate the bed shear stress. Observed differences in bed shear stress and phase lag between bed shear stress and free-stream velocity are highly sensitive to the definition of the bed position (y = b). The underestimation of turbulent stresses close to the wall is found to explain such differences when using the addition of Reynolds and viscous stresses to define both the bed shear stress and the phase lag. Regardless of the flow regime, in all experiments, boundary-layer thickness reached its maximum value at a phase near the flow reversal at the wall. Friction factors in smooth walls are better estimated using a theoretical equation first proposed by Batchelor (An introduction to fluid dynamics. Cambridge University Press, Cambridge, 1967) while the more recent empirical predictor of Pedocchi and Garcia (J Hydraul Res 47(4):438–444, 2009a) was found to be appropriate for estimating friction coefficients in the laminar-to-turbulent transition regime.

The structural characteristics of a jet heated to 425 K and emitted from a raised, circular stack into a 300 K cross-flow were studied via cross-plane stereo particle-image velocimetry measurements at multiple streamwise locations downstream of the stack exit. Similar measurements of an unheated jet at equivalent Reynolds number and similar blowing ratio provided a baseline of comparison for the heated case. Instantaneous velocity fields for the heated and unheated jets were marked by intense, small-scale vortices and only a weak indication of a counter-rotating vortex pair (CVP). Upon filtering by proper orthogonal decomposition (POD) to recover only the large-scale flow features, the imprint of the CVP was clearly discerned in the instantaneous fields. The CVP of the heated jet was stronger as well as larger and advected further into the cross-flow compared to that of the unheated jet. While this large-scale reconstruction by POD embodied 35 % of the turbulent kinetic energy, it was found that these large-scale motions captured a vast majority of the Reynolds shear stress, indicating a predominance of the CVP in this regard.

Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus.