Assessing corneal cross-linking with reverberant 3D optical coherence elastography.

SIGNIFICANCE: Corneal cross-linking (CXL) is a well-known procedure for treating certain eye disorders such as keratoconus. However, characterization of the biomechanical changes in the cornea as a result of this procedure is still under active research. Specifically, there is a clinical need for high-resolution characterization of individual corneal layers. AIM: A high-resolution elastography method in conjunction with a custom optical coherence tomography system is used to track these biomechanical changes in individual corneal layers. Pre- and post-treatment analysis for both low-dose and high-dose CXL experiments are performed. APPROACH: A recently developed elastography technique that utilizes the theory of reverberant shear wave fields, with optical coherence tomography as the modality, is applied to pig corneas ex vivo to evaluate elasticity changes associated with corneal CXL. Sets of low-dose and high-dose CXL treatments are evaluated before and after treatments with three pairs of pig corneas per experiment. RESULTS: The reverberant three-dimensional (3D) optical coherence elastography (OCE) technique can identify increases in elasticity associated with both low-dose and high-dose CXL treatments. There is a notable graphical difference between low-dose and high-dose treatments. In addition, the technique is able to identify which layers of the cornea are potentially affected by the CXL procedure and provides insight into the nonlinearity of the elasticity changes. CONCLUSIONS: The reverberant 3D OCE technique can identify depth-resolved changes in elasticity of the cornea associated with CXL procedures. This method could be translated to assess and monitor CXL efficacy in various clinical settings.

Acetonitrile cluster solvation in a cryogenic ethane-methane-propane liquid: Implications for Titan lake chemistry.

The atmosphere of Titan, Saturn's largest moon, exhibits interesting UV- and radiation-driven chemistry between nitrogen and methane, resulting in dipolar, nitrile-containing molecules. The assembly and subsequent solvation of such molecules in the alkane lakes and seas found on the moon's surface are of particular interest for investigating the possibility of prebiotic chemistry in Titan's hydrophobic seas. Here we characterize the solvation of acetonitrile, a product of Titan's atmospheric radiation chemistry tentatively detected on Titan's surface [H. B. Niemann et al., Nature 438, 779-784 (2005)], in an alkane mixture estimated to match a postulated composition of the smaller lakes during cycles of active drying and rewetting. Molecular dynamics simulations are employed to determine the potential of mean force of acetonitrile (CH3CN) clusters moving from the alkane vapor into the bulk liquid. We find that the clusters prefer the alkane liquid to the vapor and do not dissociate in the bulk liquid. This opens up the possibility that acetonitrile-based microscopic polar chemistry may be possible in the otherwise nonpolar Titan lakes.

Polymorphism and electronic structure of polyimine and its potential significance for prebiotic chemistry on Titan.

The chemistry of hydrogen cyanide (HCN) is believed to be central to the origin of life question. Contradictions between Cassini-Huygens mission measurements of the atmosphere and the surface of Saturn's moon Titan suggest that HCN-based polymers may have formed on the surface from products of atmospheric chemistry. This makes Titan a valuable "natural laboratory" for exploring potential nonterrestrial forms of prebiotic chemistry. We have used theoretical calculations to investigate the chain conformations of polyimine (pI), a polymer identified as one major component of polymerized HCN in laboratory experiments. Thanks to its flexible backbone, the polymer can exist in several different polymorphs, which are relatively close in energy. The electronic and structural variability among them is extraordinary. The band gap changes over a 3-eV range when moving from a planar sheet-like structure to increasingly coiled conformations. The primary photon absorption is predicted to occur in a window of relative transparency in Titan's atmosphere, indicating that pI could be photochemically active and drive chemistry on the surface. The thermodynamics for adding and removing HCN from pI under Titan conditions suggests that such dynamics is plausible, provided that catalysis or photochemistry is available to sufficiently lower reaction barriers. We speculate that the directionality of pI's intermolecular and intramolecular =N-H(…)N hydrogen bonds may drive the formation of partially ordered structures, some of which may synergize with photon absorption and act catalytically. Future detailed studies on proposed mechanisms and the solubility and density of the polymers will aid in the design of future missions to Titan.

Engaging novice researchers in the process and culture of science using a "Pass-the-Problem" case strategy.

Undergraduates having their first research experience frequently have little idea of what to expect. Institutions offering summer research experiences attempt to address this issue through programs that introduce students to the process and culture of science. However, didactic approaches frequently bore students who prefer more interactive sessions. We describe a "Pass-the-Problem" case study approach that engages groups of students in useful discussions about the research environment they are entering. The cases presented here include keeping a thorough laboratory notebook, balancing laboratory and personal time demands, anxiety about formal presentations, unexpected federal regulatory inspection, working in a lab with limited funds, being used as a technician rather than a researcher, frustration with failed experiments, effects of promotion and tenure on laboratory atmosphere, the importance of reading the research literature, and questioning a career in science. These cases alert students to different situations they might encounter and stimulate discussion about how to deal with them.

Advanced corneal cross-linking system with fluorescence dosimetry.

Purpose. This paper describes an advanced system that combines corneal cross-linking with riboflavin with fluorescence dosimetry, the ability to measure riboflavin diffusion within the cornea both before and during UVA treatment. Methods and Results. A corneal cross-linking system utilizing a digital micromirror device (DMD) was assembled and used to measure diffusion coefficients of 0.1% riboflavin in 20% dextran in porcine eyes. A value of (3.3 ± 0.2) × 10(-7) cm(2)/s was obtained for the stroma. Diffusion coefficients for the transepithelial formulation of 0.1% riboflavin in 0.44% saline and 0.02% BAK were also measured to be 4.7 ± 0.3 × 10(-8) cm(2)/s for epithelium only and (4.6 ± 0.4) × 10(-7) cm(2)/s for stroma only. Riboflavin consumption during a UVA treatment was also demonstrated. Conclusion. A new advanced corneal cross-linking system with fluorescence dosimetry of riboflavin has been demonstrated. It is hoped that this method may play a significant role in determining the underlying mechanisms of corneal cross-linking and assist with the development of additional riboflavin formulations. Moreover, dosimetry may prove valuable in providing a method to account for the biological differences between individuals, potentially informing cornea-specific UVA treatment doses in real time.

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice.

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.

Toward integration: from quantitative biology to mathbio-biomath?

In response to the call of BIO2010 for integrating quantitative skills into undergraduate biology education, 30 Howard Hughes Medical Institute (HHMI) Program Directors at the 2006 HHMI Program Directors Meeting established a consortium to investigate, implement, develop, and disseminate best practices resulting from the integration of math and biology. With the assistance of an HHMI-funded mini-grant, led by Karl Joplin of East Tennessee State University, and support in institutional HHMI grants at Emory and University of Delaware, these institutions held a series of summer institutes and workshops to document progress toward and address the challenges of implementing a more quantitative approach to undergraduate biology education. This report summarizes the results of the four summer institutes (2007-2010). The group developed four draft white papers, a wiki site, and a listserv. One major outcome of these meetings is this issue of CBE-Life Sciences Education, which resulted from proposals at our 2008 meeting and a January 2009 planning session. Many of the papers in this issue emerged from or were influenced by these meetings.

A transformative model for undergraduate quantitative biology education.

The BIO2010 report recommended that students in the life sciences receive a more rigorous education in mathematics and physical sciences. The University of Delaware approached this problem by (1) developing a bio-calculus section of a standard calculus course, (2) embedding quantitative activities into existing biology courses, and (3) creating a new interdisciplinary major, quantitative biology, designed for students interested in solving complex biological problems using advanced mathematical approaches. To develop the bio-calculus sections, the Department of Mathematical Sciences revised its three-semester calculus sequence to include differential equations in the first semester and, rather than using examples traditionally drawn from application domains that are most relevant to engineers, drew models and examples heavily from the life sciences. The curriculum of the B.S. degree in Quantitative Biology was designed to provide students with a solid foundation in biology, chemistry, and mathematics, with an emphasis on preparation for research careers in life sciences. Students in the program take core courses from biology, chemistry, and physics, though mathematics, as the cornerstone of all quantitative sciences, is given particular prominence. Seminars and a capstone course stress how the interplay of mathematics and biology can be used to explain complex biological systems. To initiate these academic changes required the identification of barriers and the implementation of solutions.

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a].

The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr-/-), and apolipoprotein E-deficient (Apoe-/-) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr-/- mice and greatly accelerated in Apoe-/- mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr-/- mice was similar to that in wild-type mice and was only slightly accelerated in Apoe-/- mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance. In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.

Abnormal in vivo metabolism of apoB-containing lipoproteins in human apoE deficiency.

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).

Characterization of lipoproteins from the turtle, Trachemys scripta elegans, in fasted and fed states.

The lipid and apolipoprotein composition of VLDL, IDL, LDL, HDL(2) and HDL(3) were examined in the turtle, Trachemys scripta elegans, in fasted and fed states. The lipid composition of turtle lipoproteins was very similar to their human counterparts. The major apolipoprotein found in LDL, IDL and VLDL, which has a molecular weight of approximately 550 kD, is a homologue of apoB100. The major apolipoprotein found in both HDL(2) and HDL(3), has a molecular weight of 28-kD and is homologous to human apoA-I. HDL(3) also contains a 6.5 kD protein that is homologous to apoA-II, while HDL(2) has two low molecular weight proteins of 6 kD and 7 kD which are also found on the triglyceride rich lipoproteins (TRL). The 7 kD protein is homologous to apoC-III, while the 6 kD protein has a similar size and distribution as apoC-II or apoC-I. In addition, HDL(2) also possesses a protein of 15.8 kD that has no obvious mammalian homologue. In both size and apolipoprotein composition, turtle HDL(2) resembles human HDL(2b) while turtle HDL(3) resembles human HDL(3). In the fasted state, turtles contained very little TRL. When fed a high fat diet, the amount of IDL and LDL sized particles increased significantly.

Apolipoprotein(A) expression in intracranial aneurysms.

OBJECTIVE: Elevated serum levels of lipoprotein(a), a risk factor for atherosclerosis (AS), are also associated with the presence of asymptomatic intracranial aneurysms. AS is present in some aneurysms, but its contribution to aneurysm formation and growth is unclear. Apolipoprotein(a) [apo(a)], the active moiety of lipoprotein(a), is present in atherosclerotic circle of Willis vessels but not in normal circle of Willis vessels. We wished to determine whether apo(a) is present in intracranial aneurysms independently of AS. METHODS: With a purified anti-apo(a) monoclonal antibody, aneurysms (n = 25) and feeding vessels (n = 23) were examined for apo(a) expression by immunohistochemical analysis. Circle of Willis arteries with and without AS (n = 19), cavernous angiomas (n = 5), and arteriovenous malformations (n = 6) acted as control samples. RESULTS: AS was present in 32% of aneurysms, and all of those aneurysms demonstrated mural immunopositivity for apo(a). However, aneurysms devoid of AS also demonstrated apo(a) immunopositivity. Apo(a) was demonstrated in 86% of available feeding vessels. Apo(a) deposition was not observed in cavernous angiomas but was present in arteriovenous malformations. Eleven Circle of Willis arteries (57.9%) were devoid of AS and demonstrated no apo(a) immunostaining, whereas the eight (42.1%) with AS were immunopositive for apo(a). CONCLUSION: Apo(a) expression in intracranial aneurysms may occur independently of AS. Apo(a) in feeding vessels suggests a possible role for apo(a) in early events leading to aneurysm formation. Multilayered transmural apo(a) deposition in established aneurysms suggests apo(a) involvement in aneurysm growth, possibly via cycles of injury and repair. The absence of apo(a) in cavernous angiomas suggests that such injury might be pressure-mediated.

Gene expression profiling of human diseases by serial analysis of gene expression.

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.

Microarray, SAGE and their applications to cardiovascular diseases.

The wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insights that have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area.

Anti-atherogenic effects of the acyl-CoA:cholesterol acyltransferase inhibitor, avasimibe (CI-1011), in cultured primary human macrophages.

Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to reduce atherosclerotic lesions in animals; however, the mechanism(s) for this effect remains unclear. Therefore, we used cultured primary human monocyte-derived macrophages (HMMs) to examine the effect of the ACAT inhibitor, avasimibe (CI-1011), during foam cell formation and during cholesterol efflux from established foam cells. To examine the effect of CI-1011 on foam cell development, HMMs were incubated with aggregated acetylated LDL (ag-acLDL)+/-CI-1011 for 48 h. Total cholesterol (TC) was 29% lower in HMMs incubated with ag-acLDL and CI-1011 compared with ag-acLDL (P<0.05). To determine if TC reduction was due to reduced ag-acLDL uptake by CI-1011, 125I-acLDL binding at 4 degrees C for 4 h to HMMs preincubated with acLDL or ag-acLDL, CI-1011, acLDL+CI-1011, or ag-acLDL+CI-1011 for 48 h was measured. Specific binding was 40% lower in cells preincubated with acLDL+CI-1011, 52% lower in cells preincubated with ag-acLDL+CI-1011 and 49% lower in cells preincubated with CI-1011 compared with cells preincubated with acLDL (P<0.0003). Because CI-1011 appeared to directly affect acLDL binding, 125I-acLDL (3-80 microg protein/ml) binding was done in HMMs preincubated with CI-1011 (0-10 microg/ml) for 48 h. The calculated B(max) decreased in HMMs exposed to increasing concentrations of CI-1011, suggesting that CI-1011 altered scavenger receptor function and/or number. To examine the effects of CI-1011 on cholesterol efflux from established foam cells, we first examined whether CI-1011 was cytotoxic. HMMs were preincubated with ag-acLDL for 24 h, and then radiolabeled with [14C]adenine for 2 h (time zero). The radiolabeled cells were exposed to control RPMI medium or the same medium+HDL, CI-1011, or HDL+CI-1011 for 24 h. The release of [14C]adenine into the medium was not significantly different between cells exposed to RPMI, HDL, CI-1011, or HDL+CI-1011, suggesting that CI-1011 was not cytotoxic. Foam cells exposed to RPMI and CI-1011 (1-10 microg/ml) for 48 h showed time dependent reduction in cellular TC mass, with a corresponding increase in radiolabeled unesterified cholesterol into the medium. We then asked whether CI-1011 enhanced apoE mediated cholesterol efflux. Although cellular apoE increased between 2- and 7-fold in foam cells compared to control macrophages, apoE secreted into the medium was not significantly different between cells exposed to RPMI or CI-1011. Thus, CI-1011 exerted anti-atherogenic effects by reducing TC accumulation, inhibiting acLDL binding, and by limiting lipid storage in HMMs.

Metal ions and the stereochemistry of ribozyme reactions.

Inversion of configuration at phosphorus during ribozyme-catalyzed cleavage of RNA is usually considered unequivocal proof of in-line attack, but the relevant pseudorotation diagram for formation of the 2('),3(')-cyclic phosphate shows that inversion is not inconsistent with adjacent attack as long as breakdown of the trigonal bipyramid is in-line. For the reaction to occur by adjacent attack, a normally unstable apical oxyanion in the trigonal bipyramidal intermediate would have to be stabilized. Density-functional calculations show that a metal ion such as magnesium could perform this stabilization. We conclude that the possibility of adjacent attack should not be too hastily dismissed in cases where the setup is closer to adjacent than to in-line geometry.