On-chip label-free protein analysis with downstream electrodes for direct removal of electrolysis products.

The ability to apply highly controlled electric fields within microfluidic devices is valuable as a basis for preparative and analytical processes. A challenge encountered in the context of such approaches in conductive media, including aqueous buffers, is the generation of electrolysis products at the electrode/liquid interface which can lead to contamination, perturb fluid flows and generally interfere with the measurement process. Here, we address this challenge by designing a single layer microfluidic device architecture where the electric potential is applied outside and downstream of the microfluidic device while the field is propagated back to the chip via the use of a co-flowing highly conductive electrolyte solution that forms a stable interface at the separation region of the device. The co-flowing electrolyte ensures that all the generated electrolysis products, including Joule heat and gaseous products, are flowed away from the chip without coming into contact with the analytes while the single layer fabrication process where all the structures are defined lithographically allows producing the devices in a simple yet highly reproducible manner. We demonstrate that by allowing stable and effective application of electric fields in excess of 100 V cm-1, the described platform provides the basis for rapid separation of heterogeneous mixtures of proteins and protein complexes directly in their native buffers as well as for the simultaneous quantification of their charge states. We illustrate this by probing the interactions in a mixture of an amyloid forming protein, amyloid-ß, and a molecular chaperone, Brichos, known to inhibit the process of amyloid formation. The availability of a platform for applying stable electric fields and its compatibility with single-layer soft-lithography processes opens up the possibility of separating and analysing a wide range of molecules on chip, including those with similar electrophoretic mobilities.

Twist-bend nematic liquid crystals in high magnetic fields.

We present magneto-optic measurements on two materials that form the recently discovered twist-bend nematic (N_{tb}) phase. This intriguing state of matter represents a fluid phase that is orientationally anisotropic in three directions and also exhibits translational order with periodicity several times larger than the molecular size. N_{tb} materials may also spontaneously form a visible, macroscopic stripe texture. We show that the optical stripe texture can be persistently inhibited by a magnetic field, and a 25T external magnetic field depresses the N-N_{tb} phase transition temperature by almost 1{∘}C. We propose a quantitative mechanism to account for this shift and suggest a Helfrich-Hurault-type mechanism for the optical stripe formation.

Magnetic-field-induced suppression of the amorphous blue phase.

We present magneto-optical measurements on two liquid crystals that exhibit a wide temperature-range amorphous blue phase (BPIII). Magnetic fields up to 25 T are found to suppress the onset of BPIII in both materials by almost 1 °C. This effect appears to increase nonlinearly with the field strength. The effect of high fields on established BPIIIs is also reported, in which we find significant hysteresis and very slow dynamics. Possible explanations of these results are discussed.

Optimizing gene transfer to conventional outflow cells in living mouse eyes.

The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 µl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.

Accounting for low-frequency synchrotron X-ray beam position fluctuations for dynamic visualizations.

Synchrotron X-ray radiography on beamline 05B1-1 at the Canadian Light Source Inc. was employed to study dynamic liquid water transport in the porous electrode materials of polymer electrolyte membrane fuel cells. Dynamic liquid water distributions were quantified for each radiograph in a sequence, and non-physical liquid water measurements were obtained. It was determined that the position of the beam oscillated vertically with an amplitude of ~25 µm at the sample and a frequency of ~50 mHz. In addition, the mean beam position moved linearly in the vertical direction at a rate of 0.74 µm s(-1). No evidence of horizontal oscillations was detected. In this work a technique is presented to account for the temporal and spatial dependence of synchrotron beam intensity, which resulted in a significant reduction in false water thickness. This work provides valuable insight into the treatment of radiographic time-series for capturing dynamic processes from synchrotron radiation.

Health literacy and vision-related quality of life.

BACKGROUND: Non-visual factors influence a person's vision-related quality of life (VRQoL). The purpose of this study was to assess the relationship between health literacy and VRQoL in glaucoma patients. METHODS: One hundred and ninety-five subjects with open-angle glaucoma participated in a cross-sectional patient survey and chart review. Subjects were administered a test of health literacy, an assessment of physical and mental well-being, and an assessment of VRQoL, the National Eye Institute 25-Item Visual Function Questionnaire (VFQ-25). Charts were reviewed for visual acuity and visual field results. RESULTS: In univariate analyses, older age (p<0.001), non-White race (p<0.001), worse visual acuity (p<0.001), worse visual field scores (p<0.001), lower level of education (p<0.001), worse health literacy (p<0.001) and worse score on the mental health component of the SF-12 (p = 0.005) were associated with worse VFQ-25 scores. In multivariate analyses, only older age was associated with worse total VFQ-25 scores (p<0.001), although the association between health literacy and the VFQ subscale of dependency remained significant (p = 0.04). CONCLUSIONS: Individuals with a lower health literacy do not appear to have a worse overall VRQoL compared with those with a higher literacy, but worse health literacy is associated with increased dependency.

Induction of TGF-beta1 in the trabecular meshwork under cyclic mechanical stress.

The pathophysiological mechanisms involved in the failure of the trabecular meshwork (TM) to maintain normal levels of aqueous outflow in glaucoma are not yet understood. Aberrant activation of the transforming growth factor beta-1 (TGF-beta1) pathway has been implicated in several degenerative diseases. We investigated the possibility that chronic cyclic mechanical stress that affects the TM might result in increased production of TGF-beta1. Primary cultures of TM cells subjected to cyclic mechanical stress (5% stretching, 1 cycle/sec) demonstrate a significant increase in total and biologically active secreted TGF-beta1 that was associated with activation of the TGF-beta1 promoter, measured using a recombinant adenovirus expressing the secreted reporter gene secreted alkaline phosphatase protein (SEAP) under the TGF-beta1 gene promoter (AdTGFbeta1-SEAP). Associated changes in the transcription of MMP-2, TIMP-2, and CTGF were assessed by semiquantitative PCR. Immunohistochemical analysis of TGF-beta1 in organ culture of human eyes revealed a generalized accumulation of this protein in the extracellular matrix (ECM) of the TM, while expression of the TGF-beta1 promoter, analyzed using the LacZ reporter gene, was localized in some specific cells within the outflow pathway. Induction of the TGF-beta1 promoter in organ culture was demonstrated using a novel model for cyclic mechanical stress in human perfused anterior segments infected with AdTGFbeta1-SEAP. Given the relevant physiological and pathophysiological roles of TGF-beta1, its induction after cyclic mechanical stress in the TM supports the hypothesis that this cytokine might play a significant role in the physiology of the TM, and contribute to the pathological changes of this tissue in certain forms of glaucoma.

Prevalence of myocilin mutations in adults with primary open-angle glaucoma in Ghana, West Africa.

PURPOSE: Investigators have noted that primary open-angle glaucoma (POAG) in West Africa has an earlier age of onset and appears to be more clinically severe than in the United States and Europe. Primary open-angle glaucoma patients with mutations in myocilin have a similar phenotype. Therefore, we investigated the role of mutations in myocilin in patients with POAG in a West African population. MATERIALS AND METHODS: Patients seen at the Emmanuel Eye Clinic in Accra, Ghana, were recruited for this study. Informed consent was obtained from all study patients. Glaucoma specialists from the sponsoring institution (PC, LWH, or RRA) ascertained all POAG and control patients. Age-matched unaffected controls were obtained in patients with an IOP < 22 mm Hg and normal-appearing optic nerves. PCR amplification of each of the three myocilin exons was performed. Denaturing high-performance liquid chromatography (Transgenomics Corp.) was used to detect allelic differences and samples demonstrating a mobility shift were sequenced in both directions. RESULTS: Ninety unrelated affecteds with POAG and 76 control patients were recruited. Four individuals with severe POAG were found to have novel missense mutations in exon 3. Two exhibit an Asp380Asn mutation and two an Arg342Lys mutation. These changes were not detected in 152 ethnically matched control chromosomes. Fourteen affected individuals and eight controls exhibit a translationally silent polymorphism in codon 325 (Thr325Thr). CONCLUSIONS: A total of 4.4% of patients with POAG have novel disease-associated mutations in myocilin. Mutations in myocilin appear to play a limited role in the pathogenesis of POAG in this region of West Africa.

A duplication in chromosome 4q35 is associated with hereditary benign intraepithelial dyskeratosis.

Hereditary benign intraepithelial dyskeratosis (HBID) is an autosomal dominant disorder characterized by elevated epithelial plaques on the ocular and oral mucous membranes. It has been reported primarily, but not exclusively, in individuals of American Indian heritage in North Carolina. We have examined and obtained DNA on two large families affected by HBID. Using genetic linkage analysis we have localized the HBID gene to chromosome 4 (4q35) with a peak LOD score of 8.97. Molecular analysis of these data reveals that all individuals affected with HBID in both families demonstrate the presence of three alleles for two tightly linked markers, D4S1652 and D4S2390, which map to the telomeric region of 4q35. This suggests the presence of a duplication segregating with the disease phenotype that is most likely involved in its causation.

Comparison of intraocular pressure lowering by topical and systemic carbonic anhydrase inhibitors in the rabbit.

We compared the effect on intraocular pressure (IOP) of maximal doses of a topical carbonic anhydrase inhibitor (CAI) at acidic and alkaline pH where it is maximally effective with full systemic CA inhibition in ocular normotensive New Zealand Albino rabbits. Tonometric IOP levels were measured hourly during 3 hour control period. Topical MK-417 (pKa 5.8, 8.3), a close congener of MK-507 (Dorzolamide) was given as a 1.4% solution at pH 4.5 (n=6) and pH 9.2 (n=6). MK-417 was instilled to the left eye with the right eye used as an untreated control. One hour later methazolamide was given intravenously at 10 mg/kg, a dose known to give full inhibition of the enzyme. Control IOP (mm Hg) was 19.12+/-0.50. One hour following MK-417, the left eye IOP was 13.40 +/-0.70 (pH 4.5) and 13.25+/-0.70 (pH 9.2). The right eye pressure was unchanged. Methazolamide injection at this time gave no further drop in the left eye IOP at either pH. IOP in the right eye fell to 14.00+/-0.70 so that 2 hours after methazolamide injection, the 2 eyes had the same pressure. In conclusion, topical CAI in sufficient dose and correct pH yields IOP lowering equivalent to a maximally effective dose of systemic CAI in rabbits.