Polymeric rapid prototyping for inexpensive and portable medical diagnostics

Tianchi Ma, Victoria Northrup, Andrew O. Fung, D. Moira Glerum, Christopher J. Backhouse, "Polymeric rapid prototyping for inexpensive and portable medical diagnostics", Proc. SPIE 8412, Photonics North 2012, 84120B (24 October 2012); doi: 10.1117/12.2001470. Copyright 2012, Society of Photo Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. https://doi.org/10.1117/12.2001470The advent of inexpensive CO2 laser systems has led to a wide range of demonstrations of microfabricated lab on chip systems built of acrylic. However, there has been little application of these systems to building microfluidics for DNA analysis. In this work we explore the use of CO2 laser systems for building microfluidics for DNA analysis and relate the artifacts of the fabrication technology to the performance of the system. We show that surface roughness that leads to significant constrictions in the separation channel provides an upper limit of the size of DNA that can be analysed. Below that upper limit, the resolution of the chip is strongly affected by the degree to which the separation channel is exposed to redeposited by-products of the ablation process. We show that by controlling these effects we are reliably able to discern two types of PCR product as a test representative of a real application. By being able to do this is in microfluidic devices the size of a postage stamp we have shown that we can now use CO2 laser systems for the development of extremely inexpensive diagnostic systems using a rapid prototyping approach.Natural Sciences and Engineering Research Council of CanadaTeledyne-DALS

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.bbabio.2018.03.011 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN-227415-2012)Undergraduate Research Internship (URI) Program at the University of Waterlo

Robust thermal control for CMOS-based lab-on-chip systems

The need for precise temperature control at small scales has provided a formidable challenge to the lab-on-chip community. It requires, at once, good thermal conductivity for high speed operation, good thermal isolation for low power consumption and the ability to have small (mm-scale) thermally independent regions on the same substrate. Most importantly, and, in addition to these conflicting requirements, there is a need to accurately measure the temperature of the active region without the need for device-to-device calibrations. We have developed and tested a design that enables thermal control of lab-on-chip devices atop silicon substrates in a way that could be integrated with the standard methods of mass-manufacture used in the electronics industry (i.e. CMOS). This is a significant step towards a single-chip lab-on-chip solution, one in which the microfluidics, high voltage electronics, optoelectronics, instrumentation electronics, and the world-chip interface are all integrated on a single substrate with multiple, independent, thermally-controlled regions based on active heating and passive cooling.Natural Sciences and Engineering Research Canada (NSERC)Teledyne-DALSA Semiconducto

Mutational Analysis of the Saccharomyces cerevisiae Cytochrome c Oxidase Assembly Protein Cox11p

Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Δcox11, like Δsco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure

Influence of Indium (III) Chloride on Human Dermal Fibroblast Cell Adhesion on Tantalum/Silicon Oxide Nano-Composites

Cell adhesion is an essential biological function for division, migration, signaling and tissue development. While it has been demonstrated that this cell function can be modified by using nanometer-scale surface topographic structures, it remains unknown how contaminants such as indium (III) ion might influence this specific cell behavior. Herein, the influence of indium chloride on human dermal fibroblast (GM5565) adhesion characteristics was investigated, given the frequent contact of contaminants with skin. The morphology of the adherent cells and their mitochondrial reticulum was characterized on cell culture dishes and nanopatterned surfaces by using fluorescence confocal microscopy and scanning electron microscopy. Results showed a significant proportion of cells lost their ability to align preferentially along the line axes of the nanopattern upon exposure to 3.2 mM indium chloride, with cells aligned within 10° of the pattern line axes reduced by as much as ~70%. Concurrent with the cell adhesion behaviors, the mitochondria in cells exposed to indium chloride exhibit a punctate staining that contrasts with the normal network of elongated tubular geometry seen in control cells. Our results demonstrate that exposure to indium chloride has detrimental effects on the behavior of human fibroblasts and adversely impacts their mitochondrial morphology. This shows the importance of evaluating the biological impacts of indium compounds

COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae

We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies

Mutations in COX15 Produce a Defect in the Mitochondrial Heme Biosynthetic Pathway, Causing Early-Onset Fatal Hypertrophic Cardiomyopathy

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patient's heart and fibroblast mitochondria, and levels of heme O were increased in the patient's heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%–70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency