Lumenal Surface Research Articles

Novel Descriptors of Coronary Wall Properties Measured by OCT at Baseline Are Associated with CAV-Related Intimal Thickening One Year after Heart Transplant

Purpose Cardiac Allograft Vasculopathy (CAV) is a frequent complication of heart transplantation (HTx). To help identify patients at risk of cardiac graft failure, early determination of coronary wall thickening is of major importance. Using highly automated 3D segmentation of coronary wall layers, novel indices of intimal and medial roughness are reported. Ability of these indices obtained at 1 month (1M) to predict intimal thickening 12 months (12M) after HTx was studied. Methods Lumen surface, intimal and media layers (as depicted by OCT) were identified using our multi-surface graph-based LOGISMOS segmentation method, reported earlier. Automated results were adjusted by experts utilizing our Just-Enough-Interaction approach. Intimal thickness (IT) was determined for each image frame of the OCT pullback obtained 1M and 12M after HTx. CAV risk was quantified as average OCT-frame-specific intimal thickening (IT12M - IT1M) after 1M—12M image registration. Intimal (medial) roughness IR (MR), reflecting the circumferential variability of the intimal (medial) outer surface, was computed as the average distance between the thin-plate spline fitted to the intimal (medial) surface and the regional peaks of individual surface points. As such, smooth (rough) intimal or medial surface would be represented by small (large) IR or MR values. Results Associations between frame-based IR or MR at 1M and frame-based IT progression were studied in 116 patients for which 1M and 12M OCT pullbacks (538 frames each) were acquired. Mixed effect analysis with individual patient as grouping factor was employed. IR and MR determined at 1M were strongly associated with IT progression 12M after HTx (IR: β=-9.49, p=0.004; MR: β=-9.76, p=0.001). In a patient-level analysis using Pearson's correlation test, the negative association was further confirmed between 1M roughness and IT progression in the per-patient-most-progressing 3 mm-long vessel segment (IR: R=-0.28, p=0.002; MR: R=-0.28, p=0.002). Conclusion Our new descriptors of intimal and medial roughness derived from fully 3D segmentation of coronary wall layers in OCT image pullbacks 1 month after HTx were associated with CAV-related progression of intimal thickness at 12 months. As such, IR and MR indices showed CAV-onset prediction ability as early as 1 month after HTx.

Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow Fibers Using a Microfluidic Device.

Cellular constructs having hollow tubular structures are expected to be used as artificial blood vessels. We have recently demonstrated that water-insoluble polyion complexes (PICs) were formed from water-soluble polysaccharides with opposite charges at the interface of coaxial flows, which resulted in the formation of hollow fibers. In this study, both inside- and outside-cell-laden chondroitin sulfate C (CS)/chitosan (CHI) hollow fibers were prepared by utilizing a microfluidic device and modification with cell adhesive molecules. Loading of type I collagen (COL) and surface modification with fibronectin and gelatin using layer-by-layer assembly techniques improved the adhesion and spreading of fibroblast cells to/on the surface of CS/CHI hollow fibers. On the other hand, by suspending mesenchymal stem cells (MSCs) in the core flow solution, cells were successfully loaded in the walls of the hollow fibers. As the culture time extended, cells trapped in the PIC structures constituting the wall of the hollow fibers migrated to the interface between the hollow fibers and the medium: cells adhered to and stretched "on" the lumen surfaces in the COL-loaded fibers. In contrast, for the case of unmodified hollow fibers, it was difficult for cells to adhere to the lumen surfaces. Therefore, cell aggregates were formed "in" the lumen. Results of the live/dead assay and MTT assay clearly demonstrated that MSCs possessed certain levels of cell viability and proliferated for up to 10 days, especially for the cases of COL-loaded hollow fibers. On the basis of these results, the utility of the present hollow fibers in the formation of cellular constructs corresponding to blood vessels is also discussed.

A pragmatic approach to understand peripheral artery lumen surface stiffness due to plaque heterogeneity

The goal of this study was to develop a pragmatic approach to build patient-specific models of the peripheral artery that are aware of plaque inhomogeneity. Patient-specific models using element-specific material definition (to understand the role of plaque composition) and homogeneous material definition (to understand the role of artery diameter and thickness) were automatically built from intravascular ultrasound images of three artery segments classified with low, average, and high calcification. The element-specific material models had average surface stiffness values of 0.0735, 0.0826, and 0.0973 MPa/mm, whereas the homogeneous material models had average surface stiffness values of 0.1392, 0.1276, and 0.1922 MPa/mm for low, average, and high calcification, respectively. Localization of peak lumen stiffness and differences in patient-specific average surface stiffness for homogeneous and element-specific models suggest the role of plaque composition on surface stiffness in addition to local arterial diameter and thickness.

Abstract 184: The Relationship Between Carotid Plaque on Magnetic Resonance Plaque Imaging and Time From Stroke Onset

Introduction: Previous studies report that rate of recurrent stroke, as well as the risk of carotid surgery, is highest in the early stages following cerebral infarction and decreases over time, which suggests a process of plaque healing and stabilization. The purpose of this study is to identify differences in carotid plaque components according to the time from stroke onset, using carotid magnetic resonance plaque imaging (MRPI) to evaluate the dynamic phase of plaque development following an acute cerebrovascular ischemic event.Methods and Results: MRPI on 3T scanners was obtained in 128 patients enrolled in the Carotid Artery Stenting study (MRCAS). MRCAS is a non-randomized multicenter prospective observational study to compare the plaque characteristics evaluated by MRI and ischemic complications after CAS in Japan. Among the 128 subjects, 60 presented with TIA or stroke and 68 were asymptomatic. The 60 TIA/stroke cases were classified into four groups based on the interval from the onset of symptoms to the date of carotid MRPI (Group 1: <14 days [n=25]; Group 2: 15-30 days[n=13]; and Group 3: >31 days [n=22]). The 68 asymptomatic cases were classified as Group 4. The volume of intraplaque hemorrhage (IPH), necrotic core, calcification and loose matrix was quantified on MRPI using Radiant DICOM viewer. Further, the presence of inner Loose Matrix (iLM) was identified as a high intensity area located between the vessel lumen and necrotic core or IPH on both T2WI and proton density weighted images.Patients characteristics were not significantly different among all groups. The mean volume of loose matrix in Group 3 was significantly lower than Group 1 and Group 4 (0 mm3 vs 12.3 mm3 and 15.5 mm3; p= 0.01, 0.03, respectively). The prevalence of iLM in Group 1 was higher than in Group 3 and 4 (68% vs 18.2%, 38.3% ; p<0.01, <0.01, respectively). Conclusions: The prevalence of inner Loose Matrix, located at the interface between the lumen surface and the necrotic core or IPH, is significantly higher in the early stage after stoke/TIA. This study suggests that carotid plaques undergo rapid evolution, especially adjacent to the luminal surface, after an acute cerebrovascular ischemic event.

Structure and Self-assembly of Negatively Supercharged Protein Cages

Proteins are excellent materials for constructing nano- to micro-meter sized compartments. For example, in nature, hollow spherical shells made of proteins, called protein cages, are widespread. Prominent examples include viruses, ferritins, carboxysomes, and others. Protein cages designed and engineered in the laboratory have also gained recent attention because of their potential use in synthetic biology, materials science, and medicine. Here, we show that engineered variants of lumazine synthase (LS) from Aquifex aeolicus self-assemble into porous shell-like structures, with striking size-expansion from the original dodecahedron composed of 12 pentamer subunits. Cryo-electron microscopy (EM) analysis has revealed that pentamers are the basic assembly units, although small conformational changes in each subunit lead to final expanded architectures composed of 36 and 72 pentamers. The underlying conformational changes likely arise from electrostatic repulsion between anionic residues originally introduced at the lumenal surface of the LS cage to encapsulate positively charged guest molecules. The plastic nature of the LS cage structure was also explored using a positively supercharged variant of the green fluorescent protein GFP(+36) as an assembly mediator. By controlling the favorable electrostatic interactions between the negatively charged LS cage and the positively charged mediator, multishell structures were created, as previously observed in some virus-like particles. These results highlight the potential of engineered LS cages for various future applications, including drug delivery and bioimaging.

Fast self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast cardiovascular magnetic resonance at 17.6\u2009T

Purpose4D flow cardiovascular magnetic resonance (CMR) and the assessment of wall shear stress (WSS) are non-invasive tools to study cardiovascular risks in vivo. Major limitations of conventional triggered methods are the long measurement times needed for high-resolution data sets and the necessity of stable electrocardiographic (ECG) triggering. In this work an ECG-free retrospectively synchronized method is presented that enables accelerated high-resolution measurements of 4D flow and WSS in the aortic arch of mice.Methods4D flow and WSS were measured in the aortic arch of 12-week-old wildtype C57BL/6 J mice (n = 7) with a radial 4D-phase-contrast (PC)-CMR sequence, which was validated in a flow phantom. Cardiac and respiratory motion signals were extracted from the radial CMR signal and were used for the reconstruction of 4D-flow data. Rigid motion correction and a first order B0 correction was used to improve the robustness of magnitude and velocity data.The aortic lumen was segmented semi-automatically. Temporally averaged and time-resolved WSS and oscillatory shear index (OSI) were calculated from the spatial velocity gradients at the lumen surface at 14 locations along the aortic arch. Reproducibility was tested in 3 animals and the influence of subsampling was investigated.ResultsVolume flow, cross-sectional areas, WSS and the OSI were determined in a measurement time of only 32 min. Longitudinal and circumferential WSS and radial stress were assessed at 14 analysis planes along the aortic arch. The average longitudinal, circumferential and radial stress values were 1.52 ± 0.29 N/m2, 0.28 ± 0.24 N/m2 and − 0.21 ± 0.19 N/m2, respectively. Good reproducibility of WSS values was observed.ConclusionThis work presents a robust measurement of 4D flow and WSS in mice without the need of ECG trigger signals. The retrospective approach provides fast flow quantification within 35 min and a flexible reconstruction framework.

Influence of wall shear stress and geometry on the lumen surface concentration of low density lipoprotein in a model abdominal aortic aneurysm

In this work, the correlation between the wall shear stress (WSS) and the surface concentration variation of low density lipoprotein (LDL) on the walls of an idealized abdominal aortic aneurysm (AAA) is explored. It is observed that on the walls of AAA, the WSS has a significant effect on the LDL deposition pattern and a region of low WSS does not always correspond to a locally high concentration of LDL. The correlation between the WSS and the LDL deposition is found to be linked to the local fluid-flow pattern. This is explained by conducting an analysis on simplified geometries using steady inflow conditions that produced a fluid-flow pattern similar to that in the original AAA under physiologically relevant pulsatile inflow conditions. Furthermore, the influence of geometric parameters, such as the height, the width, and the shape of the aneurysm, on the surface concentration of LDL is studied. The width of the aneurysm is found to have a strong influence on the variation of the LDL surface concentration.

Impact of Plaque Erosion on Stress/Strain and Flow Shear Stress Calculation: An OCT-Based FSI Modeling Study

Plaque erosion, one of the primary causes for coronary thrombosis, is responsible for about one third of the patients with acute coronary syndrome (ACS) [1]. Histological studies characterized the eroded plaque as a plaque with following morphological features: 1) plaque intima having direct contact with intraluminal thrombus due to the absence of endothelium or endothelial injury; 2) without rupture in the fibrous cap; 3) abundance of proteoglycans and smooth muscle cells on the luminal surface under the thrombus [2]. These characteristics has been applied in in vivo diagnosis of plaque erosion using optical coherence tomography (OCT) image technology and specific treatment strategy has also been developed for plaque erosion related ACS [3]. However, The pathogenesis mechanisms that cause plaque erosion are not fully understood [2]. It is postulated that plaque erosion has close association with biomechanical conditions in the coronary vessel [2]. In this work, a patient-specific OCT-based modeling approach was develop 3D fluid-structure interaction (FSI) models to investigate the biomechanical conditions in eroded plaque and investigate the impact of erosion-induced inflammation on plaque stress/strain conditions. Patient-specific OCT data of eroded coronary plaque were acquired from one male patient (age: 64) with frequency-domain OCT C7XR system and the Dragon Fly catheter (Lightlab Imaging/St. Jude Medical). Erosion sites were identified as the luminal surface having direct contact with intraluminal thrombus on the OCT images. Biplane X-ray angiographic data (Allura Xper FD10 System, Philips, Bothel, WA) were also acquired with the information of the location of the coronary artery stenosis and vessel curvature. OCT images were segmented manually with external elastic membrane (EEM) contour assumed to have positive remodeling ratio 1.1 for this plaque. The trailing edge of the lipid-rich necrotic core (lipid) was also assumed since it cannot be seen on the OCT images. Co-registration of OCT and angiographic data were performed to reconstruct the 3D coronary geometry. Our FSI modeling procedures with pre-shrink–stretch process and anisotropic material properties were previously developed. Blood flow was assumed to be laminar, Newtonian and incompressible, with arbitrary Lagrangian–Eulerian formulation of the Navier–Stokes equations used as the governing equations. The governing equations of the structure models included equations of motion, the nonlinear Cauchy–Green strain–displacement relation and Mooney–Rivlin material properties. To reflect tissue weakening caused by erosion-induced inflammation, the material stiffness of plaque intima at the erosion site was adjust to one tenth of un-eroded fibrous plaque tissue [2]. Three FSI models were constructed to investigate the impacts of erosion-induced inflammation and lipid component on plaque stress/strain conditions: M1, without erosion (this means plaque intima at the erosion sites were not softened) and without inclusion of lipid component; M2, with erosion but no lipid component; M3, with erosion and inclusion of lipid component. Pulsating pressure conditions were prescribed at the inlet and outlet of the vessel segment. Fluid–structure interaction boundary conditions including force balance and no-slip conditions were imposed to the fluid–structure interface between blood flow and vessel lumen surface. Results for plaque wall stress/strain (PWS/PWSn) and flow wall shear stress (WSS) from the FSI models corresponding to peak blood pressure were extracted for analysis. Table 1 summarizes the average values of the biomechanical factors both at erosion sites and fibrous cap overlaying lipid component for 3 models. By comparing M1 and M2 at the erosion sites, Mean PWS value decreases from 49.98 kPa to 18.83 kPa (62.32% decrease) while Mean PWSn value increases from 0.1231 to 0.1384 (12% increase) as the material stiffness becomes 10 times soft. At the fibrous cap, M3 (with inclusion of lipid) will elevates mean PWS and PWSn values by 48.59% and 16.09%, respectively. The impacts of erosion and lipid on flow shear stress were limited (<2%). Table 1: Average values of three biomechanical factors over erosion sites and fibrous cap overlaying lipid core. Average values of biomechanical factors over Erosion sites for 3 Models M1 M2 M3 Mean PWS (kPa) 49.9759 18.8318 18.8298 Mean PWSn 0.1231 0.1384 0.1384 Mean WSS (dyn/cm2) 101.0637 99.9383 99.4303 Average values of biomechanical factors over fibrous cap overlaying lipid for 3 Models M1 M2 M3 Mean PWS (kPa) 68.3209 68.3199 101.5173 Mean PWSn 0.0870 0.0870 0.1010 Mean WSS (dyn/cm2) 18.5260 18.4634 18.1910 To conclude, erosion-induced inflammation on blood vessel would lead to lower stress distribution but larger strain distribution, while lipid component would elevate both stress and strain conditions. This shows the influence of erosion and lipid component has impacts on stress/strain calculations which are closely related to plaque assessment.

Distribution of atherosclerotic lesions in various arteries of WHHLMI rabbits, an animal model of familial hypercholesterolemia.

In WHHLMI rabbits, arterial lesions develop spontaneously in various arteries even with standard chow. Here, we examined the development of arterial lesions in various arteries to demonstrate standard characteristics of arterial lesions in WHHLMI rabbits. For WHHLMI rabbits at 6, 12, 20, and 30 months of age, lesion areas and areas of arterial lumen surfaces were measured using image analysis software. Histopathological sections of arterial lesions were stained with elastic van Gieson staining. Arterial lesions developed around bifurcations and expanded with aging. In the aorta, atheromatous lesions were severe in the thoracic aorta but were mild in the distal part of the abdominal aorta. Carotid artery lesions progressed in the proximal region and at bifurcations, and the histopathological features were similar to those of coronary lesions. Pulmonary artery lesions contained many foam cells. Fibrous lesions were observed in the proximal and distal areas of the renal arteries, at the bifurcation of the iliac-femoral artery and mesenteric artery, and around the anastomosis of vertebral arteries. Lesions in the celiac artery contained foam cells and/or lipid droplets within fibrous lesions. In a pair of right and left arteries, the arterial lesions tended to progress more in the right artery. Gender did not affect analysis of arterial lesions. In conclusion, the arterial lesions expanded from bifurcations, and the morphological features of the arterial lesions varied depending on the type of artery. These results serve as reference data for arterial lesions in studies using WHHLMI rabbits.

Tubularized urethral reconstruction using a prevascularized capsular tissue prelaminated with buccal mucosa graft in a rabbit model.

Tubularized graft urethroplasty fails largely because of inadequate graft take. Prefabrication of buccal mucosa lined flap has theoretical indications for constructing neourethra with an independent blood supply. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate an axial vascularized buccal mucosa-lined flap for tubularized urethral reconstruction in a rabbit model was tested. The experiments were performed in three stages. First, silicone tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Next, buccal mucosa grafts were transplanted to the newly formed capsular tissue supplied by the axial vessel for buccal mucosa-lined flap prefabrication. Then, circumferential urethral defects were created and repaired by buccal mucosa graft (Group 1), capsule flap (Group 2) and prefabricated capsule buccal mucosa composite flap (Group 3). With retrograde urethrography, no rabbits in Group 1 maintained a wide urethral caliber. In Group 2, the discontinued epithelial layer regenerated at 1 month, and the constructed neourethra narrowed even though the lumen surface formed intact urothelial cells at 3 months. In Group 3, buccal mucosa formed the lining in the neourethra and kept a wide urethral caliber for 3 months. The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication. The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for circumferential urethral replacement.

Secondary Fill Minimizes Gutter Size in Chimney EVAS Configurations In Vitro

Purpose: To investigate in an in vitro model if secondary endobag filling can reduce gutter size during chimney endovascular aneurysm sealing (chEVAS). Materials and Methods: Nellix EVAS systems were deployed in 2 silicone juxtarenal aneurysm models with suprarenal aortic diameters of 19 and 24 mm. Four configurations were tested: EVAS with 6-mm balloon-expandable (BE) or self-expanding (SE) chimney grafts (CGs) in the renal branches of both models. Balloons were inflated simultaneously in the CGs and main endografts during primary and secondary endobag filling and polymer curing. Computed tomography (CT) was performed immediately after the primary and secondary fills. Cross-sectional lumen areas were measured on the CT images to calculate gutter volumes and percent change. CG compression was calculated as the reduction in lumen surface area measured perpendicular to the central lumen line. The largest gutter volume and highest compression were presented per CG configuration per model. Results: Secondary endobag filling reduced the largest gutter volumes from 99.4 to 73.1 mm3 (13.2% change) and 84.2 to 72.0 mm3 (27.6% change) in the BECG configurations and from 67.2 to 44.0 mm3 (34.5% change) and 92.7 to 82.3 mm3 (11.2% change) in the SECG configurations in the 19- and 24-mm models, respectively. Secondary endobag filling increased CG compression in 6 of 8 configurations. BECG compression changed by −0.2% and 5.4% and by −1.0% and 0.4% in the 19- and 24-mm models, respectively. SECG compression changed by 10.2% and 16.0% and by 7.2% and 7.3% in the 19- and 24-mm models, respectively. Conclusion: Secondary endobag filling reduced paragraft gutters; however, this technique did not obliterate them. Increased CG compression and prolonged renal ischemia time should be considered if secondary endobag filling is used.

Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila

In yeast, the Atg2-Atg18 complex regulates Atg9 recycling from phagophore assembly site during autophagy; their function in higher eukaryotes remains largely unknown. In a targeted screening in Drosophila melanogaster, we show that Mef2-GAL4-RNAi-mediated knockdown of Atg2, Atg9 or Atg18 in the heart and indirect flight muscles led to shortened healthspan (declined locomotive function) and lifespan. These flies displayed an accelerated age-dependent loss of cardiac function along with cardiac hypertrophy (increased heart tube wall thickness) and structural abnormality (distortion of the lumen surface). Using the Mef2-GAL4-MitoTimer mitochondrial reporter system and transmission electron microscopy, we observed significant elongation of mitochondria and reduced number of lysosome-targeted autophagosomes containing mitochondria in the heart tube but exaggerated mitochondrial fragmentation and reduced mitochondrial density in indirect flight muscles. These findings provide the first direct evidence of the importance of Atg2-Atg18/Atg9 autophagy complex in the maintenance of mitochondrial integrity and, regulation of heart and muscle functions in Drosophila, raising the possibility of augmenting Atg2-Atg18/Atg9 activity in promoting mitochondrial health and, muscle and heart function.

Electrochemical fabrication of a biomimetic elastin-containing bi-layered scaffold for vascular tissue engineering

Biomimetic tissue-engineered vascular grafts (TEVGs) have immense potential to replace diseased small-diameter arteries (<4 mm) for the treatment of cardiovascular diseases. However, biomimetic approaches developed thus far only partially recapitulate the physicochemical properties of the native vessel. While it is feasible to fabricate scaffolds that are compositionally similar to native vessels (collagen and insoluble elastic matrix) using freeze-drying, these scaffolds do not mimic the aligned topography of collagen and elastic fibers found in native vessels. Extrusion-based scaffolds exhibit anisotropic collagen orientation but these scaffolds are compositionally dissimilar (cannot incorporate insoluble elastic matrix). In this study, an electrochemical fabrication technique was employed to develop a biomimetic elastin-containing bi-layered collagen scaffold which is compositionally and structurally similar to native vessels and the effect of insoluble elastin incorporation on scaffold mechanics and smooth muscle cell (SMC) response was investigated. Further, the functionality of human umbilical vein endothelial cells (HUVECs) on the scaffold lumen surface was assessed via immunofluorescence. Results showed that incorporation of insoluble elastin maintained the overall collagen alignment within electrochemically aligned collagen (ELAC) fibers and this underlying aligned topography can direct cellular orientation. Ring test results showed that circumferential orientation of ELAC fibers significantly improved scaffold mechanics. Real-time PCR revealed that the expression of α-smooth muscle actin (Acta2) and myosin heavy chain (MyhII) was significantly higher on elastin containing scaffolds suggesting that the presence of insoluble elastin can promote contractility in SMCs. Further, mechanical properties of the scaffolds significantly improved post-culture indicating the presence of a mature cell-synthesized and remodeled matrix. Finally, HUVECs expressed functional markers on collagen lumen scaffolds. In conclusion, electrochemical fabrication is a viable method for the generation of a functional biomimetic TEVG with the potential to be used in bypass surgery.

ROCK-nmMyoII, Notch and Neurog3 gene-dosage link epithelial morphogenesis with cell fate in the pancreatic endocrine-progenitor niche.

During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed Neurog3LO state, progenitors become endocrine committed via upregulation of Neurog3 We find that the Neurog3LO to Neurog3HI transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and Neurog3 transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through Neurog3-expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and Neurog3 upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, Neurog3 gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche.

Structure, mechanism, and regulation of the chloroplast ATP synthase

INTRODUCTION Green plant chloroplasts convert light into chemical energy, and adenosine triphosphate (ATP) generated by photosynthesis is the prime source of biologically useful energy on the planet. Plants produce ATP by the chloroplast F 1 F o ATP synthase (cF 1 F o ), a macromolecular machine par excellence, driven by the electrochemical proton gradient across the photosynthetic membrane. It consists of 26 protein subunits, 17 of them wholly or partly membrane-embedded. ATP synthesis in the hydrophilic α 3 β 3 head (cF 1 ) is powered by the cF o rotary motor in the membrane. cF o contains a rotor ring of 14 c subunits, each with a conserved protonatable glutamate. Subunit a conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and e transmits the torque from the F o motor to the catalytic cF 1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b′, and δ act as a stator to prevent unproductive rotation of cF 1 with cF o . All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF 1 F o has a redox switch that inhibits adenosine triphosphatase (ATPase) activity in the dark. RATIONALE Understanding the molecular mechanisms of this elaborate nanomachine requires detailed structures of the whole complex, ideally at atomic resolution. Because of the dynamic nature of this membrane protein complex, crystallization has been difficult and no high-resolution structure of an entire, functional ATP synthase is available. We reconstituted cF 1 F o from spinach chloroplasts into lipid nanodiscs and determined its structure by cryo–electron microscopy (cryo-EM). Cryo-EM is the ideal technique for this study because it can deliver high-resolution structures of large, dynamic macromolecular assemblies that adopt a mixture of conformational states. RESULTS We present the cryo-EM structure of the intact cF 1 F o ATP synthase in lipid nanodiscs at a resolution of 2.9 A (cF 1 ) to 3.4 A (cF o ). In the cF 1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase head binds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α helices of the peripheral stalk subunits b and b′ clamp the integral membrane subunit a in its position next to the c-ring rotor, thus connecting cF 1 to cF o . Subunit γ has an L-shaped double hairpin with a redox sensor that can form a disulfide bond and a chock that blocks rotation to avoid wasteful ATP hydrolysis at night. Protons are translocated through access routes in subunit a in all rotary ATPases. We observe a hydrophilic channel on the lumenal surface that connects to the glutamate residues on the c-ring rotor that carry protons for an almost full rotation before releasing them into the stroma through another hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane. CONCLUSION We observe three cF 1 F o conformations, each with the central rotor stalled in a different position. Ring rotation is unexpectedly divided into three unequal steps. The peripheral stalk may thus act like an elastic spring, evening out the different energy contributions of each step. The features of ATP synthase nanomachines are remarkably similar in chloroplasts and mitochondria, considering their evolutionary distance of a billion years or more.

A Patient-Specific Study Investigating the Relation between Coronary Hemodynamics and Neo-Intimal Thickening after Bifurcation Stenting with a Polymeric Bioresorbable Scaffold

We present an application of a validated reconstruction methodology for the comparison between patient-specific hemodynamics and neo-intimal thickening at nine months from the intervention. (1) Background: Coronary bifurcation stenting alters the vessel geometry, influencing the local hemodynamics. The evaluation of wall shear stress (WSS) relies on the application of computational fluid dynamics to model its distribution along the coronary tree. The endothelium actively responds to WSS, which triggers eventual cell proliferation to cover the stent struts. (2) Methods: Baseline optical coherence tomography and angiographic data were combined to reconstruct a patient-specific coronary bifurcation with an implanted bioresorbable scaffold and to simulate the hemodynamics. Results were linked with the neo-intimal thickening after nine months from the intervention. (3) Results: Blood velocity patterns were disrupted at the bifurcation due to the presence of the stent. It was observed that 55.6% of the scaffolded lumen surface was exposed to values of time-averaged WSS lower than 0.4 Pa. Follow-up images showed a luminal narrowing of 19% in the main branch. There was also a complete coverage in 99% of struts. (4) Conclusions: This approach provided valuable complementary information that might improve the clinical outcomes in this subset of coronary diseases.

Relationship Between Central Venous Catheter Protein Adsorption and Water Infused Surface Protection Mechanisms.

Central venous catheters (CVCs) are implanted in the majority of dialysis patients despite increased patient risk due to thrombotic occlusion and biofilm formation. Current solutions remain ineffective at preventing these complications and treatment options are limited and often harmful. We present further analysis of the previously proposed water infused surface protection (WISP) technology, an active method to reduce protein adsorption and effectively disrupt adsorbed protein sheaths on the inner surface of CVCs. A WISP CVC is modeled by a hollow fiber membrane (HFM) in a benchtop device which continuously infuses a saline solution across the membrane wall into the blood flow, creating a blood-free boundary layer at the lumen surface. Total protein adsorption is measured under various experimental conditions to further test WISP performance. The WISP device shows reduced protein adsorption as blood and WISP flow rates increase (P < 0.040) with up to a 96% reduction in adsorption over the no WISP condition. When heparin is added to the WISP flow, protein adsorption (0.097[+0.035/-0.055] µg/mm2 ) is reduced when compared to both bolus administration and nondoped WISP, 0.406(+0.056/-0.065) µg/mm2 (P = 0.001) and 0.191 (+0.076/-0.126) (P = 0.029), respectively. Additionally, when heparinized WISP is applied to a preadsorbed protein layer, 0.375(+0.114/-0.164) µg/mm2 , it displays the ability to reduce the previously-adsorbed protein, 0.186(+0.058/-0.084) µg/mm2 (P = 0.0012), suggesting aptitude for intermittent treatments. The WISP technology not only shows the ability to reduce protein adsorption, but also the ability to remove preadsorbed material by effectively delivering drugs to the point of adsorption; functionalities that could greatly improve clinical outcomes.

Rational Engineering of a Designed Protein Cage for siRNA Delivery.

Oligonucleotide therapeutics have transformative potential in modern medicine but are poor drug candidates in themselves unless fitted with compensatory carrier systems. We describe a simple approach to transform a designed porous protein cage into a nucleic acid delivery vehicle. By introducing arginine mutations to the lumenal surface, a positively supercharged capsule is created, which can encapsidate oligonucleotides in vitro with high binding affinity. We demonstrate that the siRNA-loaded cage is taken up by mammalian cells and releases its cargo to induce RNAi and knockdown gene expression. These general concepts could also be applied to alternative scaffold designs, expediting the development of artificial protein cages toward delivery applications.

Cell-layer dependent adhesion differences in wood bonds

The performance of wood adhesives depends on its internal cohesion and the adhesion to the wood surface which is structurally and chemically heterogeneous as it may be formed by different micro-surface, i.e. the compound middle lamella, the secondary cell wall layer, or the lumen surface. This study introduces an experimental protocol to directly evaluate adhesion to each of these micro-surfaces individually. Two wood fiber types presumed to differ in their surface chemistry are used as substrate. The results demonstrate that melamine-urea-formaldehyde adhesive shows excellent adhesion irrespective of the fiber type or micro-surface. While the same is the case for adhesion between untreated wood fiber surfaces and one component polyurethane, higher relative work of indentation between the lignin-rich compound middle lamella surface of medium density fiberboard fiber and one component polyurethane was observed. This observation is corroborated by a reduced polar character of this micro-surface as shown by X-ray photoelectron spectroscopy.

Measuring magnetosomal pH of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using pH-sensitive fluorescent proteins.

Magnetotactic bacteria synthesize uniform-sized and regularly shaped magnetic nanoparticles in their organelles termed magnetosomes. Homeostasis of the magnetosome lumen must be maintained for its role accomplishment. Here, we developed a method to estimate the pH of a single living cell of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using a pH-sensitive fluorescent protein E2GFP. Using the pH measurement, we estimated that the cytoplasmic pH was approximately 7.6 and periplasmic pH was approximately 7.2. Moreover, we estimated pH in the magnetosome lumen and cytoplasmic surface using fusion proteins of E2GFP and magnetosome-associated proteins. The pH in the magnetosome lumen increased during the exponential growth phase when magnetotactic bacteria actively synthesize magnetite crystals, whereas pH at the magnetosome surface was not affected by the growth stage. This live-cell pH measurement method will help for understanding magnetosome pH homeostasis to reveal molecular mechanisms of magnetite biomineralization in the bacterial organelle.