Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements.

An inexpensive digital microfluidic (DMF) chip was fabricated by screen-printing electrodes on a sheet of polyimide. This device was manually integrated with surface acoustic wave nebulization (SAWN) MS to conduct hydrogen/deuterium exchange (HDX) of peptides. The HDX experiment was performed by DMF mixing of one aqueous droplet of angiotensin II with a second containing various concentrations of D2O. Subsequently, the degree of HDX was measured immediately by SAWN-MS. As expected for a small peptide, the isotopically resolved mass spectrum for angiotensin revealed that maximum deuterium exchange was achieved using 50% D2O. Additionally, using SAWN-MS alone, the global HDX kinetics of ubiquitin were found to be similar to published NMR data and back exchange rates for the uncooled apparatus using high inlet capillary temperatures was less than 6%.

Feeding in billfishes: inferring the role of the rostrum from a biomechanical standpoint.

Perhaps the most striking feature of billfishes is the extreme elongation of the premaxillary bones forming their rostra. Surprisingly, the exact role of this structure in feeding is still controversial. The goal of this study is to investigate the use of the rostrum from a functional, biomechanical and morphological standpoint to ultimately infer its possible role during feeding. Using beam theory, experimental and theoretical loading tests were performed on the rostra from two morphologically different billfish, the blue marlin (Makaira nigricans) and the swordfish (Xiphias gladius). Two loading regimes were applied (dorsoventral and lateral) to simulate possible striking behaviors. Histological samples and material properties of the rostra were obtained along their lengths to further characterize structure and mechanical performance. Intraspecific results show similar stress distributions for most regions of the rostra, suggesting that this structure may be designed to withstand continuous loadings with no particular region of stress concentration. Although material stiffness increased distally, flexural stiffness increased proximally owing to higher second moment of area. The blue marlin rostrum was stiffer and resisted considerably higher loads for both loading planes compared with that of the swordfish. However, when a continuous load along the rostrum was considered, simulating the rostrum swinging through the water, swordfish exhibited lower stress and drag during lateral loading. Our combined results suggest that the swordfish rostrum is suited for lateral swiping to incapacitate their prey, whereas the blue marlin rostrum is better suited to strike prey from a wider variety of directions.

Identification of novel suggestive loci for high-grade myopia in Polish families.

PURPOSE: Myopia is the most common human eye disorder with complex genetic and environmental causes. To date, several myopia loci have been identified in families of different geographic origin. However, no causative gene(s) have yet been identified. The aim of this study was the characterization of Polish families with high-grade myopia, including genetic analysis. METHODS: Forty-two multiplex Polish families with non-syndromic high-grade myopia participated in the study. All family members underwent detailed ophthalmic examination and high-grade myopia was defined as ≤-6.0 diopters (D) based on the spherical refractive error. A genome-wide single nucleotide polymorphism (SNP)-based high-density linkage scan was performed using Affymetrix Human SNP Array 6.0 on a selected family (HM-32) with multiple affected individuals. RESULTS: Nonparametric linkage analysis identified three novel loci in family HM-32 at chromosome 7p22.1-7p21.1 ([NPL] 8.26; p=0.006), chromosome 7p12.3-7p11.2 ([NPL] 8.23; p=0.006), and chromosome 12p12.3-12p12.1 ([NPL] 8.02; p=0.006), respectively. The effect of linkage disequilibrium on linkage due to dense SNP map was addressed by systematically pruning SNPs from the linkage panel. CONCLUSIONS: Haplotype analysis with informative crossovers in affected individuals defined a 12.2; 10.9; and 9.5 Mb genomic regions for high-grade myopia spanned between SNP markers rs11977885/rs10950639, rs11770622/rs9719399, and rs4763417/rs10842388 on chromosomes 7p22.1-7p21.1, 7p12.3-7p11.2, and 12p12.3-12p12.1, respectively.

Localization of a gene for keratoconus to a 5.6-Mb interval on 13q32.

PURPOSE: Keratoconus (KTCN) is a noninflammatory thinning and anterior protrusion of the cornea that results in steepening and distortion of the cornea, altered refractive powers, and reduced visual acuity. Several loci responsible for a familial form of KTCN have been mapped, however; no mutations in any genes have been identified for any of these loci. There is also evidence that VSX1 and SOD1 may be involved in the etiology of KTCN. The purpose of this study was to verify the available data and to identify a new keratoconus susceptibility locus. METHODS: KTCN without other ocular or systemic features was diagnosed in 18 families. VSX1 and SOD1 sequencing was performed on affected individuals and control subjects. Genomewide linkage analysis was then performed in all families using polymorphic microsatellite markers with an average spacing of 5 cM. Next, single-nucleotide polymorphism (SNP) arrays, fluorescence in situ hybridization (FISH) analysis, and a comparative genomic hybridization array were used in one family to assess a candidate region on 13q32. RESULTS: All previously reported KTCN loci were excluded. VSX1 and SOD1 were sequenced, and no potentially functional variants were found. One KTCN family yielded a maximum multipoint parametric LOD score of 4.1 and multipoint nonparametric linkage (NPL) LOD score of 3.2. Multipoint linkage and haplotype analysis narrowed the locus to a 5.6-Mb region between the SNPs rs9516572 and rs3825523 on 13q32. CONCLUSIONS: The results exclude VSX1 and SOD1 as potential disease-causing genes in these families and localize a novel gene for keratoconus to a 5.6-Mb interval on 13q32.

Primary congenital glaucoma and Rieger's anomaly: extended haplotypes reveal founder effects for eight distinct CYP1B1 mutations.

PURPOSE: Mutations in the cytochrome P450 1B1 (CYP1B1) gene are a frequent cause of primary congenital glaucoma (PCG) in different ethnic groups. Cytochrome P450 proteins are monooxygenases, which catalyze many reactions involved in the metabolism of drugs as well as steroids and other lipids. The repeated occurence of several mutations in various ethnic groups raises the question if founder effects or mutation-prone sites in CYP1B1 are the cause for this observation. METHODS: A total of 30 individuals (26 PCG patients, three Rieger's anomaly patients, and one variant carrier), presenting 17 variants in CYP1B1 (15 mutations and two variations) were included in our study. We sequenced the entire genomic region of CYP1B1 and analyzed microsatellites flanking the gene in all individuals and constructed haplotypes for all variations using a combination of single nucleotide polymorphisms and microsatellites. RESULTS: For the CYP1B1 genomic region, we identified five extended haplotypes associated with 17 variations. These haplotypes were complemented with microsatellite information from the region surrounding this gene. A total of eight CYP1B1 mutations were found more than once, each of them presenting one identical haplotype in different individuals. Six mutations were represented in different ethnic groups. CONCLUSIONS: Our results confirm founder effects for most of CYP1B1 mutations. Most of these mutations must have occurred as unique events in the past.