Orthogonal polarization spectroscopy to detect mesenteric hypoperfusion

OBJECTIVE: Orthogonal polarization spectral (OPS) imaging is used to assess mucosal microcirculation. We tested sensitivity and variability of OPS in the assessment of mesenteric blood flow (Q (sma)) reduction. SETTING: University Animal Laboratory. INTERVENTIONS: In eight pigs, Q (sma) was reduced in steps of 15% from baseline; five animals served as controls. Jejunal mucosal microcirculatory blood flow was recorded with OPS and laser Doppler flowmetry at each step. OPS data from each period were collected and randomly ordered. Samples from each period were individually chosen by two blinded investigators and quantified [capillary density (number of vessels crossing predefined lines), number of perfused villi] after agreement on the methodology. MEASUREMENT AND RESULTS: Interobserver coefficient of variation (CV) for capillary density from samples representing the same flow condition was 0.34 (0.04-1.41) and intraobserver CV was 0.10 (0.02-0.61). Only one investigator observed a decrease in capillary density [to 62% (48-82%) of baseline values at 45% Q (sma) reduction; P = 0.011], but comparisons with controls never revealed significant differences. In contrast, reduction in perfused villi was detected by both investigators at 75% of mesenteric blood flow reduction. Laser Doppler flow revealed heterogeneous microcirculatory perfusion. CONCLUSIONS: Assessment of capillary density did not reveal differences between animals with and without Q (sma) reduction, and evaluation of perfused villi revealed blood flow reduction only when Q (sma) was very low. Potential explanations are blood flow redistribution and heterogeneity, and suboptimal contrast of OPS images. Despite agreement on the method of analysis, interobserver differences in the quantification of vessel density on gut mucosa using OPS are high.

Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.

Nucleic acid sensing by an orthogonal reporter system based on homo-DNA

We have developed an assay for single strand DNA or RNA detection which is based on the homo-DNA templated Staudinger reduction of the profluorophore rhodamine-azide. The assay is based on a three component system, consisting of a homo-DNA/DNA hybrid probe, a set of homo-DNA reporter strands and the target DNA or RNA. We present two different formats of the assay (Omega probe and linear probe) in which the linear probe was found to perform best with catalytic turnover of the reporter strands (TON: 8) and a match/mismatch discrimination of up to 19. The advantage of this system is that the reporting (homo-DNA) and sensing (DNA) domain are decoupled from each other since the two pairing systems are bioorthogonal. This allows independent optimization of either domain which may lead to higher selectivity in in vivo imaging.

Surface-acoustic-wave counterflow micropumps for on-chip liquid motion control in two-dimensional microchannel arrays

Fully controlled liquid injection and flow in hydrophobic polydimethylsiloxane (PDMS) two-dimensional microchannel arrays based on on-chip integrated, low-voltage-driven micropumps are demonstrated. Our architecture exploits the surface-acoustic-wave (SAW) induced counterflow mechanism and the effect of nebulization anisotropies at crossing areas owing to lateral propagating SAWs. We show that by selectively exciting single or multiple SAWs, fluids can be drawn from their reservoirs and moved towards selected positions of a microchannel grid. Splitting of the main liquid flow is also demonstrated by exploiting multiple SAW beams. As a demonstrator, we show simultaneous filling of two orthogonal microchannels. The present results show that SAW micropumps are good candidates for truly integrated on-chip fluidic networks allowing liquid control in arbitrarily shaped two-dimensional microchannel arrays.

A Portable Image Overlay Projection Device for Computer-Aided Open Liver Surgery

Image overlay projection is a form of augmented reality that allows surgeons to view underlying anatomical structures directly on the patient surface. It improves intuitiveness of computer-aided surgery by removing the need for sight diversion between the patient and a display screen and has been reported to assist in 3-D understanding of anatomical structures and the identification of target and critical structures. Challenges in the development of image overlay technologies for surgery remain in the projection setup. Calibration, patient registration, view direction, and projection obstruction remain unsolved limitations to image overlay techniques. In this paper, we propose a novel, portable, and handheld-navigated image overlay device based on miniature laser projection technology that allows images of 3-D patient-specific models to be projected directly onto the organ surface intraoperatively without the need for intrusive hardware around the surgical site. The device can be integrated into a navigation system, thereby exploiting existing patient registration and model generation solutions. The position of the device is tracked by the navigation system’s position sensor and used to project geometrically correct images from any position within the workspace of the navigation system. The projector was calibrated using modified camera calibration techniques and images for projection are rendered using a virtual camera defined by the projectors extrinsic parameters. Verification of the device’s projection accuracy concluded a mean projection error of 1.3 mm. Visibility testing of the projection performed on pig liver tissue found the device suitable for the display of anatomical structures on the organ surface. The feasibility of use within the surgical workflow was assessed during open liver surgery. We show that the device could be quickly and unobtrusively deployed within the sterile environment.

Spatial and functional relationships between air conduits and blood capillaries in the pulmonary gas exchange tissue of adult and developing chickens

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung.

A triplet spike-timing–dependent plasticity model generalizes the Bienenstock–Cooper–Munro rule to higher-order spatiotemporal correlations

Synaptic strength depresses for low and potentiates for high activation of the postsynaptic neuron. This feature is a key property of the Bienenstock–Cooper–Munro (BCM) synaptic learning rule, which has been shown to maximize the selectivity of the postsynaptic neuron, and thereby offers a possible explanation for experience-dependent cortical plasticity such as orientation selectivity. However, the BCM framework is rate-based and a significant amount of recent work has shown that synaptic plasticity also depends on the precise timing of presynaptic and postsynaptic spikes. Here we consider a triplet model of spike-timing–dependent plasticity (STDP) that depends on the interactions of three precisely timed spikes. Triplet STDP has been shown to describe plasticity experiments that the classical STDP rule, based on pairs of spikes, has failed to capture. In the case of rate-based patterns, we show a tight correspondence between the triplet STDP rule and the BCM rule. We analytically demonstrate the selectivity property of the triplet STDP rule for orthogonal inputs and perform numerical simulations for nonorthogonal inputs. Moreover, in contrast to BCM, we show that triplet STDP can also induce selectivity for input patterns consisting of higher-order spatiotemporal correlations, which exist in natural stimuli and have been measured in the brain. We show that this sensitivity to higher-order correlations can be used to develop direction and speed selectivity.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Sugar modification as a means to increase the biological performance of oligonucleotides

Modification of the ribose unit in DNA and RNA profoundly influences the self-rcognition and the biological properties of the nucleic acids. Conformational restriction of the ribose units, as in LNA and tricyclo-DNA, has been identified as a powerful tool to increase DNA and RNA affinity as well as biological stability and antisense properties. Apart from that sugar modified DNA analogues, as homo-DNA, have shown to be orthogonal base-pairing systems which by virtue of non-crosscommunicating with the natural nucleic acids open novel applications in biotechnology.