Cloning, Sequencing, and Characterization of Luciola italica Luciferase

The characteristic yellow-green light of a firefly is the result of a multi-step reaction catalyzed by the luciferase enzyme. This enzyme has many applications in the biomedical field and ongoing work is being done to alter its properties to better fit these applications. The purpose of this project was to clone the Luciola italica luciferase cDNA and to express, purify and fully characterize the corresponding bioluminescence-catalyzing enzyme in hopes of obtaining novel bioluminescent materials. Fireflies were collected in the countryside of Bologna, Italy, flash frozen in liquid nitrogen and total RNA was extracted from the firefly lanterns. The L. italica luciferase cDNA was successfully cloned by RT-PCR using a gene-specific primer set based on the DNA sequence of the Eastern European Luciola mingrelica luciferase gene. The L. italica cDNA was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Initial characterization of the enzyme showed that the L. italica protein exhibits bioluminescent activity similar in intensity to the common North American Photinus pyralis luciferase; however it produces light that is slightly red-shifted (having maximum emission at 564 nm). By steady state kinetics analysis, the L. italica Km for LH2 was found to be 0.095 mM, and that of P. pyralis is 0.015 mM. On the converse, both enzymes had similar Km values for Mg-ATP (0.160 mM for P. pyralis and 0.180 mM for L. italica). The L. italica enzyme was found to sustain its light in the visible region for a longer period of time than the P. pyralis enzyme. Phylogenetic analysis showed that the L. italica luciferase gene has 95.8% and 95.6% amino acid sequence identity to the Hotaria unmunsana (Korea) and Hotaria parvula (Japan) luciferase proteins, respectively. The processes that were used to clone the Luciola italica luciferase gene, characterize the protein, and optimize protein growth conditions ar

Barriers to exercise in people with Parkinson disease

BACKGROUND: Exercise is known to reduce disability and improve quality of life in people with Parkinson disease (PD). Although barriers to exercise have been studied in older adults, barriers in people with chronic progressive neurological diseases, such as PD, are not well defined. OBJECTIVE: The purpose of this study was to identify perceived barriers to exercise in people with PD. DESIGN: The study had a cross-sectional design. METHODS: People who had PD, dwelled in the community, and were at stage 2.4 on the Hoehn and Yahr scale participated in this cross-sectional study (N=260; mean age=67.7 years). Participants were divided into an exercise group (n=164) and a nonexercise group (n=96). Participants self-administered the barriers subscale of the Physical Fitness and Exercise Activity Levels of Older Adults Scale, endorsing or denying specific barriers to exercise participation. Multivariate logistic regression analysis was used to examine the contribution of each barrier to exercise behavior, and odds ratios were reported. RESULTS: Three barriers were retained in the multivariate regression model. The nonexercise group had significantly greater odds of endorsing low outcome expectation (ie, the participants did not expect to derive benefit from exercise) (odds ratio [OR]=3.93, 95% confidence interval [CI]=2.08–7.42), lack of time (OR=3.36, 95% CI=1.55–7.29), and fear of falling (OR=2.35, 95% CI=1.17–4.71) than the exercise group. LIMITATIONS: The cross-sectional nature of this study limited the ability to make causal inferences. CONCLUSIONS: Low outcome expectation from exercise, lack of time to exercise, and fear of falling appear to be important perceived barriers to engaging in exercise in people who have PD, are ambulatory, and dwell in the community. These may be important issues for physical therapists to target in people who have PD and do not exercise regularly. The efficacy of intervention strategies to facilitate exercise adherence in people with PD requires further investigation

Identification of a Rare Coding Variant in Complement 3 Associated with Age-related Macular Degeneration

Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry matched controls revealed two large-effect rare variants; previously described R1210C in the CFH gene (fcase = 0.51%, fcontrol = 0.02%, OR = 23.11), and newly identified K155Q in the C3 gene (fcase = 1.06%, fcontrol = 0.39%, OR = 2.68). The variants suggest decreased inhibition of C3 by Factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Genome-wide association meta-analysis of corneal curvature identifies novel loci and shared genetic influences across axial length and refractive error

Abstract: Corneal curvature, a highly heritable trait, is a key clinical endophenotype for myopia - a major cause of visual impairment and blindness in the world. Here we present a trans-ethnic meta-analysis of corneal curvature GWAS in 44,042 individuals of Caucasian and Asian with replication in 88,218 UK Biobank data. We identified 47 loci (of which 26 are novel), with population-specific signals as well as shared signals across ethnicities. Some identified variants showed precise scaling in corneal curvature and eye elongation (i.e. axial length) to maintain eyes in emmetropia (i.e. HDAC11/FBLN2 rs2630445, RBP3 rs11204213); others exhibited association with myopia with little pleiotropic effects on eye elongation. Implicated genes are involved in extracellular matrix organization, developmental process for body and eye, connective tissue cartilage and glycosylation protein activities. Our study provides insights into population-specific novel genes for corneal curvature, and their pleiotropic effect in regulating eye size or conferring susceptibility to myopia