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DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies.
© 2016 The Protein Society.
PURPOSE - To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics.
METHODS - Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards.
RESULTS - Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ.
CONCLUSIONS - Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.
PURPOSE - To quantify regional changes of conventional outflow caused by ab interno trabeculectomy (AIT).
METHODS - Gonioscopic, plasma-mediated AIT was established in enucleated pig eyes. We developed a program to automatically quantify outflow changes (R, package eye-canalogram, github.com) using a fluorescent tracer reperfusion technique. Trabecular meshwork (TM) ablation was demonstrated with fluorescent spheres in six eyes before formal outflow quantification with two-dye reperfusion canalograms in six additional eyes. Eyes were perfused with a central, intracameral needle at 15 mm Hg. Canalograms and histology were correlated for each eye.
RESULTS - The pig eye provided a model with high similarity to AIT in human patients. Histology indicated ablation of TM and unroofing of most Schlemm's canal segments. Spheres highlighted additional circumferential and radial outflow beyond the immediate area of ablation. Differential canalograms showed that AIT caused an increase of outflow of 17 ± 5-fold inferonasally, 14 ± 3-fold superonasally, and also an increase in the opposite quadrants with a 2 ± 1-fold increase superotemporally, and 3 ± 3 inferotemporally. Perilimbal specific flow image analysis showed an accelerated nasal filling with an additional perilimbal flow direction into adjacent quadrants.
CONCLUSIONS - A quantitative, differential canalography technique was developed that allows us to quantify supraphysiological outflow enhancement by AIT.
BACKGROUND AND OBJECTIVE - Optic nerve sheath fenestration is an established procedure for relief of potentially damaging overpressure on the optic nerve resulting from idiopathic intracranial hypertension. Prior work showed that a mid-IR free-electron laser could be delivered endoscopically and used to produce an effective fenestration. This study evaluates the efficacy of fenestration using a table-top mid-IR source based on a Raman-shifted alexandrite (RSA) laser.
STUDY DESIGN/MATERIALS AND METHODS - Porcine optic nerves were ablated using light from an RSA laser at wavelengths of 6.09, 6.27, and 6.43 μm and pulse energies up to 3 mJ using both free-space and endoscopic beam delivery through 250-μm I.D. hollow-glass waveguides. Waveguide transmission was characterized, ablation thresholds and etch rates were measured, and the efficacy of endoscopic fenestration was evaluated for ex vivo exposures using both optical coherence tomography and histological analysis.
RESULTS - Using endoscopic delivery, the RSA laser can effectively fenestrate porcine optic nerves. Performance was optimized at a wavelength of 6.09 μm and delivered pulse energies of 0.5-0.8 mJ (requiring 1.5-2.5 mJ to be incident on the waveguide). Under these conditions, the ablation threshold fluence was 0.8 ± 0.2 J/cm(2) , the ablation rate was 1-4 μm/pulse, and the margins of ablation craters showed little evidence of thermal or mechanical damage. Nonetheless, nominally identical exposures yielded highly variable ablation rates. This led to fenestrations that ranged from too deep to too shallow-either damaging the underlying optic nerve or requiring additional exposure to cut fully through the sheath. Of 48 excised nerves subjected to fenestration at 6.09 μm, 16 ex vivo fenestrations were judged as good, 23 as too deep, and 9 as too shallow.
CONCLUSIONS - Mid-IR pulses from the RSA laser, propagated through a flexible hollow waveguide, are capable of cutting through porcine optic nerve sheaths in surgically relevant times with reasonable accuracy and low collateral damage. This can be accomplished at wavelengths of 6.09 or 6.27 μm, with 6.09 μm slightly preferred. The depth of ex vivo fenestrations was difficult to control, but excised nerves lack a sufficient layer of cerebrospinal fluid that would provide an additional margin of safety in actual patients.
© 2015 Wiley Periodicals, Inc.
PURPOSE - Organ-level registration is critical to image-guided therapy in soft tissue. This is especially important in organs such as the kidney which can freely move. We have developed a method for registration that combines three-dimensional locations from a holographic conoscope with an endoscopically obtained textured surface. By combining these data sources clear decisions as to the tissue from which the points arise can be made.
METHODS - By localizing the conoscope's laser dot in the endoscopic space, we register the textured surface to the cloud of conoscopic points. This allows the cloud of points to be filtered for only those arising from the kidney surface. Once a valid cloud is obtained we can use standard surface registration techniques to perform the image-space to physical-space registration. Since our methods use two distinct data sources we test for spatial accuracy and characterize temporal effects in phantoms, ex vivo porcine and human kidneys. In addition we use an industrial robot to provide controlled motion and positioning for characterizing temporal effects.
RESULTS - Our initial surface acquisitions are hand-held. This means that we take approximately 55 s to acquire a surface. At that rate we see no temporal effects due to acquisition synchronization or probe speed. Our surface registrations were able to find applied targets with submillimeter target registration errors.
CONCLUSION - The results showed that the textured surfaces could be reconstructed with submillimetric mean registration errors. While this paper focuses on kidney applications, this method could be applied to any anatomical structures where a line of sight can be created via open or minimally invasive surgical techniques.
Gastric motility is coordinated by bio-electrical events known as slow waves. Abnormalities in slow waves are linked to major functional and motility disorders. In recent years, the use of high-resolution (HR) recordings have provided a unique view of spatiotemporal activation profiles of normal and dysrhythmic slow wave activity. To date, in vivo studies of gastric slow wave activity have primarily focused on the activation phase of the slow wave event. In this study, the recovery phase of slow waves was investigated through the use of HR recording techniques. The recovery phase of the slow wave event was detected through the use of the signal derivative, computed via a wavelet transform. The activation to recovery interval (ARi) metric was computed as a difference between the recovery time and activation time. The detection method was validated with synthetic slow wave signals of varying morphologies with the addition of synthetic ventilator and high frequency noise. The methods was then applied to HR experimental porcine gastric slow wave recordings. Ventilator noise more than 10% of the slow wave amplitude affected the estimation of the ARi metric. Signal to noise ratio below 3 dB affected the ARi metric, but with minor deviation in accuracy. Experimental ARi values ranged from 3.7-4.7 s from three data sets, with significant differences across them.
Silicon nanopore membranes (SNMs) with compact geometry and uniform pore size distribution have demonstrated a remarkable capacity for hemofiltration. These advantages could potentially be used for hemodialysis. Here, we present an initial evaluation of the SNM's mechanical robustness, diffusive clearance, and hemocompatibility in a parallel plate configuration. Mechanical robustness of the SNM was demonstrated by exposing membranes to high flows (200 ml/min) and pressures (1,448 mm Hg). Diffusive clearance was performed in an albumin solution and whole blood with blood and dialysate flow rates of 25 ml/min. Hemocompatibility was evaluated using scanning electron microscopy and immunohistochemistry after 4 hours in an extracorporeal porcine model. The pressure drop across the flow cell was 4.6 mm Hg at 200 ml/min. Mechanical testing showed that SNM could withstand up to 775.7 mm Hg without fracture. Urea clearance did not show an appreciable decline in blood versus albumin solution. Extracorporeal studies showed blood was successfully driven via the arterial-venous pressure differential without thrombus formation. Bare silicon showed increased cell adhesion with a 4.1-fold increase and 1.8-fold increase over polyethylene glycol (PEG)-coated surfaces for tissue plasminogen factor (t-PA) and platelet adhesion (CD41), respectively. These initial results warrant further design and development of a fully scaled SNM-based parallel plate dialyzer for renal replacement therapy.
This study reports the initial in vivo use of a combined radiofrequency ablation and cryo-anchoring (RFC) catheter as an alternative to surgical mitral valve (MV) leaflet resection. Radiofrequency ablation thermally shrinks enlarged collagenous tissues, providing an alternative to leaflet resection, and cryo-anchoring provides reversible attachment of a catheter to freely mobile MV leaflets. Excised porcine MVs (n = 9) were tested in a left heart flow simulator to establish treatment efficacy criteria. Resected leaflet area was quantified by tracking markers on the leaflet surface, and leaflet length reductions were directly measured on echocardiography. Leaflet area decreased by 38 ± 2.7%, and leaflet length decreased by 9.2 ± 1.8% following RFC catheter treatment. The RFC catheter was then tested acutely in healthy pigs (n = 5) under epicardial echocardiographic guidance, open-chest without cardiopulmonary bypass, using mid-ventricular free wall access. Leaflet length was quantified using echocardiography. Quantitative assessment of MV leaflet length revealed that leaflet resection was successful in 4 of 5 pigs, with a leaflet length reduction of 13.3 ± 4.6%. Histological, mechanical, and gross pathological findings also confirmed that RFC catheter treatment was efficacious. The RFC catheter significantly reduces MV leaflet size in an acute animal model, providing a possible percutaneous alternative to surgical leaflet resection.
Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a 'nutrient-sensitized' chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.
The filling of wound cavities with new tissue is a challenge. We previously reported on the physical properties and wound healing kinetics of prefabricated, gas-blown polyurethane (PUR) scaffolds in rat and porcine excisional wounds. To address the capability of this material to fill complex wound cavities, this study examined the in vitro and in vivo reparative characteristics of injected PUR scaffolds employing a sucrose porogen. Using the porcine excisional wound model, we compared reparative outcomes to both preformed and injected scaffolds as well as untreated wounds at 9, 13, and 30 days after scaffold placement. Both injected and preformed scaffolds delayed wound contraction by 19% at 9 days and 12% at 13 days compared to nontreated wounds. This stenting effect proved transient since both formulations degraded by day 30. Both types of scaffolds significantly inhibited the undesirable alignment of collagen and fibroblasts through day 13. Injected scaffolds were highly compatible with sentinel cellular events of normal wound repair cell proliferation, apoptosis, and blood vessel density. The present study provides further evidence that either injected or preformed PUR scaffolds facilitate wound healing, support tissue infiltration and matrix production, delay wound contraction, and reduce scarring in a clinically relevant animal model, which underscores their potential utility as a void-filling platform for large cutaneous defects. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1679-1690, 2016.
© 2015 Wiley Periodicals, Inc.