A fibrin enhanced thrombosis model for medical devices operating at low shear regimes or large surface areas.

Over the past decade, much of the development of computational models of device-related thrombosis has focused on platelet activity. While those models have been successful in predicting thrombus formation in medical devices operating at high shear rates (> 5000 s-1), they cannot be directly applied to low-shear devices, such as blood oxygenators and catheters, where emerging information suggest that fibrin formation is the predominant mechanism of clotting and platelet activity plays a secondary role. In the current work, we augment an existing platelet-based model of thrombosis with a partial model of the coagulation cascade that includes contact activation of factor XII and fibrin production. To calibrate the model, we simulate a backward-facing-step flow channel that has been extensively characterized in-vitro. Next, we perform blood perfusion experiments through a microfluidic chamber mimicking a hollow fiber membrane oxygenator and validate the model against these observations. The simulation results closely match the time evolution of the thrombus height and length in the backward-facing-step experiment. Application of the model to the microfluidic hollow fiber bundle chamber capture both gross features such as the increasing clotting trend towards the outlet of the chamber, as well as finer local features such as the structure of fibrin around individual hollow fibers. Our results are in line with recent findings that suggest fibrin production, through contact activation of factor XII, drives the thrombus formation in medical devices operating at low shear rates with large surface area to volume ratios.

Establishment and evaluation of a rat model of extracorporeal membrane oxygenation (ECMO) thrombosis using a 3D-printed mock-oxygenator.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) research using large animals requires a significant amount of resources, slowing down the development of new means of ECMO anticoagulation. Therefore, this study developed and evaluated a new rat ECMO model using a 3D-printed mock-oxygenator. METHODS: The circuit consisted of tubing, a 3D-printed mock-oxygenator, and a roller pump. The mock-oxygenator was designed to simulate the geometry and blood flow patterns of the fiber bundle in full-scale oxygenators but with a low (2.5 mL) priming volume. Rats were placed on arteriovenous ECMO at a 1.9 mL/min flow rate at two different heparin doses (n = 3 each): low (15 IU/kg/h for eight hours) versus high (50 IU/kg/h for one hour followed by 25 IU/kg/h for seven hours). The experiment continued for eight hours or until the mock-oxygenator failed. The mock-oxygenator was considered to have failed when its blood flow resistance reached three times its baseline resistance. RESULTS: During ECMO, rats maintained near-normal mean arterial pressure and arterial blood gases with minimal hemodilution. The mock-oxygenator thrombus weight was significantly different (p < 0.05) between the low (0.02 ± 0.006 g) and high (0.003 ± 0.001 g) heparin delivery groups, and blood flow resistance was also larger in the low anticoagulation group. CONCLUSIONS: This model is a simple, inexpensive system for investigating new anticoagulation agents for ECMO and provides low and high levels of anticoagulation that can serve as control groups for future studies.

Autophagic Upregulation Is Cytoprotective in Ischemia/Reperfusion-Injured Retina and Retinal Progenitor Cells.

The cytoprotective versus cytotoxic role of macroautophagy in ocular ischemia/reperfusion injuries remains controversial and its effects under hyperglycemia are unclear. We investigated the involvement of autophagy in in vitro and in vivo normoglycemic and hyperglycemic models of retinal ischemia/reperfusion injury. Retinal ischemia (2 h) and reperfusion (2 or 22 h) was induced in wild-type and type I diabetic Ins2Akita/+ mice using a middle cerebral artery occlusion model. R28 retinal precursor cells were subjected to CoCl2-induced hypoxia with or without autophagic inhibitor NH4Cl. Autophagic regulation during ischemia/reperfusion was assessed through immunohistochemical detection and Western blotting of microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal associated membrane protein 1 (LAMP1). Effect of autophagic inhibition on cell viability and morphology under hypoxic conditions was also evaluated. Upregulation of autophagic markers in the inner retinae was seen after two hours reperfusion, with tapering of the response following 22 h of reperfusion in vivo. LC3-II turnover assays confirmed an increase in autophagic flux in our hypoxic in vitro model. Pharmacological autophagic inhibition under hypoxic conditions decreased cell survival and induced structural changes not demonstrated with autophagic inhibition alone. Yet no statistically significant different autophagic responses in ischemia/reperfusion injuries were seen between the two glycemic states.

ABO blood group and procoagulant factors: the hypercoagulation hypothesis ABO and Procoagulant Factors.

BACKGROUND: The correlation between procoagulant levels-factor VIII (FVIII), von Willebrand factor (vWF), and fibrinogen-and risk of thrombosis has been well documented in adult populations. We hypothesize that interaction of passively transferred isoagglutinins in premature neonates with a compromised immune system may trigger an immune response that can target the immature gastrointestinal tract. The objective of this study is to evaluate if there are procoagulant level differences in preterm newborns stratified by ABO blood group. METHODS: VWF, FVIII, and fibrinogen levels were analyzed in neonates ≤32 weeks and/or birthweight ≤1500 g over the first 6 weeks of life. Demographic, blood type, and transfusion data were collected. RESULTS: Elevations in vWF and FVIII were found to be statistically significant in the third week of life in non-O neonates vs. type O neonates. FVIII was also found to be significantly elevated in week 1. Transfused neonates also showed elevations between weeks 0 and 3. CONCLUSION: There appears to be a time-dependent variation in procoagulant factor levels in preterm newborns. Although the clinical significance remains unclear, prothrombotic factors vWF and FVIII are significantly higher in non-O blood-type preterm neonates in the third week of life.

Comparison of Adherence to the 2013 ACC/AHA Cholesterol Guideline in a Teaching Versus Nonteaching Outpatient Clinic.

BACKGROUND: Little information is available regarding prescribers' adherence rate to the 2013 American College of Cardiology (ACC)/American Heart Association (AHA) cholesterol guideline, especially that from a teaching versus a nonteaching setting. OBJECTIVES: We aim to evaluate adherence rates to the 2013 ACC/AHA cholesterol guideline in a teaching versus a nonteaching practice site. In addition, the impact of a pharmacist-led seminar on adherence rate to the guideline was assessed. METHODS: This study is a 2-part retrospective chart review. Part 1 consists of patients who were initiated on statin therapy between December 2013 and November 2014. Patients were analyzed to determine if they received concordant statin therapy as recommended by the guideline. For the second part, we evaluated the impact of a seminar on the adherence rate to the guideline. RESULTS: Of the 325 patients who received a statin prescription, 233 were included in the study. Prescriber adherence to the guideline was 42.9%, which was significantly lower than the 65.8% observed in a study previously conducted at a teaching outpatient clinic ( P < 0.0001). For the second part of our study, prescriber adherence to the guideline 3 months before the pharmacist-led seminar was 53.5%, and this adherence rate remained virtually unchanged at 54.2% at 3 months after the educational session. CONCLUSION: The overall adherence rate to the 2013 ACC/AHA cholesterol guideline from this nonteaching outpatient clinic was significantly lower than that previously observed in a teaching outpatient clinic. The single pharmacist-led seminar did not significantly affect prescribers' adherence rate to the guideline.

Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium.

The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function in vivo, yet current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications.

Exchange protein activated by cAMP 1 (Epac1)-deficient mice develop ß-cell dysfunction and metabolic syndrome.

Previously, exchange protein directly activated by cAMP 2 (Epac2) and PKA were known to play a role in glucose-stimulated insulin secretion (GSIS) by pancreatic ß cells. The present study shows that Epac1 mRNA is also expressed by ß cells. Therefore, we generated mice and embryonic stem (ES) cells with deletion of the Epac1 gene to define its role in ß-cell biology and metabolism. The homozygous Epac1-knockout (Epac1(-/-)) mice developed impaired glucose tolerance and GSIS with deranged islet cytoarchitecture, which was confirmed by isolated islets from adult Epac1(-/-) mice. Moreover, Epac1(-/-) mice developed more severe hyperglycemia with increased ß-cell apoptosis and insulitis after multiple low-dose streptozotocin (MLDS; 40 mg/kg) treatment than Epac1(+/+) mice. Interestingly, Epac1(-/-) mice also showed metabolic defects, including increased respiratory exchange ratio (RER) and plasma triglyceride (TG), and more severe diet-induced obesity with insulin resistance, which may contributed to ß-cell dysfunction. However, islets differentiated from Epac1(-/-) ES cells showed insulin secretion defect, reduced Glut2 and PDX-1 expression, and abolished GLP-1-stimulated PCNA induction, suggesting a role of Epac1 in ß-cell function. The current study provides in vitro and in vivo evidence that Epac1 has an important role in GSIS of ß cells and phenotype resembling metabolic syndrome.

Angiotensin converting enzyme inhibitors and Alzheimer disease in the presence of the apolipoprotein E4 allele.

OBJECTIVE: The effect of angiotensin converting enzyme (ACE) inhibitors on Alzheimer disease (AD) remains unclear, with conflicting results reported. We studied the interaction of the Apolipoprotein E (ApoE) genotype and ACE inhibitors on AD. METHODS: This was a cross-sectional study of homebound elderly with an AD diagnosis and documentation of medications taken. ApoE genotype was determined. RESULTS: A total of 355 subjects with status on ApoE alleles and cognitive diagnoses were studied. The average age (mean ± SD) of this population was 73.3 ± 8.3 years old, and 73% were female. Cross-sectionally, there was no difference in the number of AD cases between ApoE4 carriers and ApoE4 non-carriers or between ACE inhibitor users and non-users in the homebound elderly. ApoE4 carriers treated with ACE inhibitors, however, had more diagnoses of AD compared with those who did not have the treatment (28% versus 6%, p = 0.01) or ApoE4 non-carriers treated with an ACE inhibitor (28% versus 10%, p = 0.03). ACE inhibitor use was associated with AD diagnosis only in the presence of an E4 allele. Using multivariate logistic regression analysis, we found that in diagnosed AD cases there was a significant interaction between ApoE4 and ACE inhibitor use (odds ratio: 20.85; 95% confidence interval: 3.08-140.95; p = 0.002) after adjusting for age, sex, ethnicity, and education. CONCLUSION: The effects of ACE inhibitors on AD may be different depending on ApoE genotype. A prospective study is needed to determine whether ACE inhibitor use accelerates or poorly delays AD development in ApoE4 carriers compared with ApoE4 non-carriers.

Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations.

Carney-Stratakis syndrome, an inherited condition predisposing affected individuals to gastrointestinal stromal tumor (GIST) and paraganglioma, is caused by germline mutations in succinate dehydrogenase (SDH) subunits B, C, or D, leading to dysfunction of complex II of the electron transport chain. We evaluated the role of defective cellular respiration in sporadic GIST lacking mutations in KIT or PDGFRA (WT). Thirty-four patients with WT GIST without a personal or family history of paraganglioma were tested for SDH germline mutations. WT GISTs lacking demonstrable SDH genetic inactivation were evaluated for SDHB expression by immunohistochemistry and Western blotting and for complex II activity. For comparison, SDHB expression was also determined in KIT mutant and neurofibromatosis-1-associated GIST, and complex II activity was also measured in SDH-deficient paraganglioma and KIT mutant GIST; 4 of 34 patients (12%) with WT GIST without a personal or family history of paraganglioma had germline mutations in SDHB or SDHC. WT GISTs lacking somatic mutations or deletions in SDH subunits had either complete loss of or substantial reduction in SDHB protein expression, whereas most KIT mutant GISTs had strong SDHB expression. Complex II activity was substantially decreased in WT GISTs. WT GISTs, particularly those in younger patients, have defects in SDH mitochondrial complex II, and in a subset of these patients, GIST seems to arise from germline-inactivating SDH mutations. Testing for germline mutations in SDH is recommended in patients with WT GIST. These findings highlight a potential central role of SDH dysregulation in WT GIST oncogenesis.

Effect of Artificial Lung Fiber Bundle Geometric Design on Micro- and Macro-scale Clot Formation.

The hollow fiber membrane bundle is the functional component of artificial lungs, transferring oxygen and carbon dioxide to and from the blood. It is also the primary location of blood clot formation and propagation in these devices. The geometric design of fiber bundles is defined by a narrow range of parameters that determine gas exchange efficiency and blood flow resistance, such as fiber packing density, path length, and frontal area. However, these parameters also affect thrombosis. This study investigated the effect of these parameters on clot formation using 3-D printed flow chambers that mimic the geometry and blood flow patterns of fiber bundles. Hollow fibers were represented by an array of vertical micro-rods (380 micron diameter) arranged with varying packing densities (40, 50, and 60%) and path lengths (2 and 4 cm). Blood was pumped through the device corresponding to three mean blood flow velocities (16, 20, and 25 cm/min). Results showed that (1) clot formation decreases dramatically with decreasing packing density and increasing blood flow velocity, (2) clot formation at the outlet of fiber bundle enhances deposition upstream, and consequently (3) greater path length provides more clot-free fiber surface area for gas exchange than a shorter path length. These results can be used to create less thrombogenic, more efficient artificial lung designs. Translational Impact Sentence: Fiber bundle parameters, such as decreased packing density, increased blood flow velocity, and a longer path length, can be used to design a less thrombogenic, more efficient artificial lung to extend functionality.

Pilot Testing of a Lightweight, Pulmonary Assist System in an Ambulatory Sheep Model of Destination Therapy Respiratory Support.

A new, lightweight (2.3 kg), ambulatory pulmonary assist system (PAS) underwent preliminary evaluation in ambulatory sheep. The PAS was purposefully designed for long-term extracorporeal respiratory support for chronic lung disease and utilizes a novel, small (0.9 m 2 surface area) gas exchanger, the pulmonary assist device, with a modified Heart Assist 5 pump fitting in a small wearable pack. Prototype PAS were attached to two sheep in venovenous configuration for 7 and 14 days, evaluating ability to remain thrombus free; maintain gas exchange and blood flow resistance; avoid biocompatibility-related complications while allowing safe ambulation. The PAS achieved 1.56 L/min of flow at 10.8 kRPM with a 24 Fr cannula in sheep one and 2.0 L/min at 10.5 kRPM with a 28 Fr cannula in sheep 2 without significant change. Both sheep walked freely, demonstrating the first application of truly ambulatory ECMO in sheep. While in vitro testing evaluated PAS oxygen transfer rates of 104.6 ml/min at 2 L/min blood flow, oxygen transfer rates averaged 60.6 ml/min and 70.6 ml/min in studies 1 and 2, due to average hemoglobin concentrations lower than humans (8.9 and 10.5 g/dl, respectively). The presented cases support uncomplicated ambulation using the PAS.

Factor XII Silencing Using siRNA Prevents Thrombus Formation in a Rat Model of Extracorporeal Life Support.

Heparin anticoagulation increases the bleeding risk during extracorporeal life support (ECLS). This study determined whether factor XII (FXII) silencing using short interfering RNA (siRNA) can provide ECLS circuit anticoagulation without bleeding. Adult male, Sprague-Dawley rats were randomized to four groups (n = 3 each) based on anticoagulant: (1) no anticoagulant, (2) heparin, (3) FXII siRNA, or (4) nontargeting siRNA. Heparin was administered intravenously before and during ECLS. FXII or nontargeting siRNA were administered intravenously 3 days before the initiation of ECLS via lipidoid nanoparticles. The rats were placed on pumped, arteriovenous ECLS for 8 hours or until the blood flow resistance reached three times its baseline resistance. Without anticoagulant, mock-oxygenator resistance tripled within 7 ± 2 minutes. The resistance in the FXII siRNA group did not increase for 8 hours. There were no significant differences in resistance or mock-oxygenator thrombus volume between the FXII siRNA and the heparin groups. However, the bleeding time in the FXII siRNA group (3.4 ± 0.6 minutes) was significantly shorter than that in the heparin group (5.5 ± 0.5 minutes, p < 0.05). FXII silencing using siRNA provided simpler anticoagulation of ECLS circuits with reduced bleeding time as compared to heparin. http://links.lww.com/ASAIO/A937.

Evaluating Pharmacist-Led Heart Failure Transitions of Care Clinic: Impact of Analytic Approach on Readmission Rate Endpoints.

Studies evaluating pharmacist-led transitions of care (TOC) services for heart failure patients reported profound decreases in hospital readmissions. Most studies restricted their analysis to clinic attendees (as-treated analysis), which can introduce selection and immortal time bias. In this study, we evaluated the impact of including only clinic attendees vs all clinic referrals in assessing the effectiveness of a pharmacist-led heart failure transitions of care (PharmD HF TOC) clinic program on 30-day readmissions. This is a retrospective, observational study of patients discharged from a heart failure hospitalization at a large urban academic medical center from August 2016 to December 2018. Primary exposure was the provision of a PharmD HF TOC clinic appointment in the intent-to-treat analysis and the attendance of the clinic in the as-treated analysis. Primary outcome was all-cause readmissions within 30 days of discharge. There were 766 and 1015 patients included in the as-treated and intent-to-treat analyses, respectively. In the as-treated analysis, 30-day all-cause readmissions were significantly lower in the intervention group compared to the control group (12.4% vs 19.6%, P = 0.018). In contrast, the intent-to-treat analysis did not reveal a significant difference in 30-day all-cause readmissions between the intervention group and the control group (18.2% vs 19.6%, P = 0.643). Pharmacist-led heart failure TOC program is associated with a reduction in 30-day all-cause readmissions only when restricting the analysis to clinic attendees. Future studies evaluating the effectiveness of post-discharge TOC services need to carefully consider the biases inherent in the evaluation methods employed.

H5N1 influenza virus-induced mediators upregulate RIG-I in uninfected cells by paracrine effects contributing to amplified cytokine cascades.

Highly pathogenic avian influenza H5N1 viruses cause severe disease in humans, and dysregulation of cytokine responses is believed to contribute to the pathogenesis of human H5N1 disease. However, mechanisms leading to the increased induction of proinflammatory cytokines by H5N1 viruses are poorly understood. We show that the innate sensing receptor RIG-I is involved in interferon regulatory factor 3 (IRF3), NF-κB nuclear translocation, p38 activation, and the subsequent interferon (IFN) ß, IFN-λ1, and tumor necrosis factor α induction during H5N1 infection. Soluble mediators from H5N1-infected human macrophages upregulate RIG-I, MDA5, and TLR3 to much higher levels than those from seasonal H1N1 in uninfected human macrophages and alveolar epithelial cells via paracrine IFNAR1/JAK but not IFN-λ receptor signaling. Compared with H1N1 virus-induced mediators, H5N1 mediators markedly enhance the cytokine response to PolyIC and to both seasonal and H5N1 virus infection in a RIG-I-dependent manner. Thus, sensitizing neighboring cells by upregulation of RIG-I contributes to the amplified cytokine cascades during H5N1 infection.

Inclisiran: Small Interfering Ribonucleic Acid Injectable for the Treatment of Hyperlipidemia.

Elevated plasma lipid levels, especially low-density lipoprotein, are correlated with atherosclerotic cardiovascular disease (ASCVD) and increased risk of ischemic heart disease and stroke. Statins are first-line agents for reducing low-density lipoprotein cholesterol (LDL-C) and the risk of major cardiovascular events, but patients with a genetic susceptibility or established ASCVD oftentimes remain subtherapeutic on statin therapy alone. Biotechnological advancements in medication therapy have led to the development of inclisiran, a recently approved twice-yearly injectable agent to help patients with heterozygous familial hypercholesterolemia and clinical ASCVD on a maximally tolerated statin to reach LDL-C targets. Inclisiran has demonstrated robust LDL-C reduction in clinical trials in combination with a favorable safety profile; however, the effect on cardiovascular clinical outcomes still remains under evaluation.

Computational simulations of the 4D micro-circulatory network in zebrafish tail amputation and regeneration.

Wall shear stress (WSS) contributes to the mechanotransduction underlying microvascular development and regeneration. Using computational fluid dynamics, we elucidated the interplay between WSS and vascular remodelling in a zebrafish model of tail amputation and regeneration. The transgenic Tg (fli1:eGFP; Gata1:ds-red) zebrafish line was used to track the three-dimensional fluorescently labelled vascular endothelium for post-image segmentation and reconstruction of the fluid domain. Particle image velocimetry was used to validate the blood flow. Following amputation to the dorsal aorta and posterior cardinal vein (PCV), vasoconstriction developed in the dorsal longitudinal anastomotic vessel (DLAV) along with increased WSS in the proximal segmental vessels (SVs) from amputation. Angiogenesis ensued at the tips of the amputated DLAV and PCV where WSS was minimal. At 2 days post amputation (dpa), vasodilation occurred in a pair of SVs proximal to amputation, followed by increased blood flow and WSS; however, in the SVs distal to amputation, WSS normalized to the baseline. At 3 dpa, the blood flow increased in the arterial SV proximal to amputation and through anastomosis with DLAV formed a loop with PCV. Thus, our in silico modelling revealed the interplay between WSS and microvascular adaptation to changes in WSS and blood flow to restore microcirculation following tail amputation.

Exacerbated VEGF up-regulation accompanies diabetes-aggravated hemorrhage in mice after experimental cerebral ischemia and delayed reperfusion.

Reperfusion therapy is the preferred treatment for ischemic stroke, but is hindered by its short treatment window, especially in patients with diabetes whose reperfusion after prolonged ischemia is often accompanied by exacerbated hemorrhage. The mechanisms underlying exacerbated hemorrhage are not fully understood. This study aimed to identify this mechanism by inducing prolonged 2-hour transient intraluminal middle cerebral artery occlusion in diabetic Ins2Akita/+ mice to mimic patients with diabetes undergoing delayed mechanical thrombectomy. The results showed that at as early as 2 hours after reperfusion, Ins2Akita/+ mice exhibited rapid development of neurological deficits, increased infarct and hemorrhagic transformation, together with exacerbated down-regulation of tight-junction protein ZO-1 and up-regulation of blood-brain barrier-disrupting matrix metallopeptidase 2 and matrix metallopeptidase 9 when compared with normoglycemic Ins2+/+ mice. This indicated that diabetes led to the rapid compromise of vessel integrity immediately after reperfusion, and consequently earlier death and further aggravation of hemorrhagic transformation 22 hours after reperfusion. This observation was associated with earlier and stronger up-regulation of pro-angiogenic vascular endothelial growth factor (VEGF) and its downstream phospho-Erk1/2 at 2 hours after reperfusion, which was suggestive of premature angiogenesis induced by early VEGF up-regulation, resulting in rapid vessel disintegration in diabetic stroke. Endoplasmic reticulum stress-related pro-apoptotic C/EBP homologous protein was overexpressed in challenged Ins2Akita/+ mice, which suggests that the exacerbated VEGF up-regulation may be caused by overwhelming endoplasmic reticulum stress under diabetic conditions. In conclusion, the results mimicked complications in patients with diabetes undergoing delayed mechanical thrombectomy, and diabetes-induced accelerated VEGF up-regulation is likely to underlie exacerbated hemorrhagic transformation. Thus, suppression of the VEGF pathway could be a potential approach to allow reperfusion therapy in patients with diabetic stroke beyond the current treatment window. Experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong [CULATR 3834-15 (approval date January 5, 2016); 3977-16 (approval date April 13, 2016); and 4666-18 (approval date March 29, 2018)].

Autosomal recessive LRP1-related syndrome featuring cardiopulmonary dysfunction, bone dysmorphology, and corneal clouding.

We provide the first study of two siblings with a novel autosomal recessive LRP1-related syndrome identified by rapid genome sequencing and overlapping multiple genetic models. The patients presented with respiratory distress, congenital heart defects, hypotonia, dysmorphology, and unique findings, including corneal clouding and ascites. Both siblings had compound heterozygous damaging variants, c.11420G > C (p.Cys3807Ser) and c.12407T > G (p.Val4136Gly) in LRP1, in which segregation analysis helped dismiss additional variants of interest. LRP1 analysis using multiple human/mouse data sets reveals a correlation to patient phenotypes of Peters plus syndrome with additional severe cardiomyopathy and blood vessel development complications linked to neural crest cells.

In vitro self-assembly of tailorable nanotubes from a simple protein building block.

We demonstrate a method for generating discretely structured protein nanotubes from the simple ring-shaped building block, homohexameric Hcp1 from Pseudomonas aeruginosa. Our design exploited the observation that the crystal lattice of Hcp1 contains rings stacked in a repeating head-to-tail pattern. High-resolution detail of the ring-ring interface allowed the selection of sites for specific cysteine mutations capable of engaging in disulfide bond formation across rings, thereby generating stable Hcp1 nanotubes. Protein nanotubes containing up to 25 subunits ( approximately 100 nm in length) were self-assembled under simple conditions. Furthermore, we demonstrate that the tube ends and interior can be independently and specifically functionalized to generate nanocapsules.