Identification and replication of three novel myopia common susceptibility gene loci on chromosome 3q26 using linkage and linkage disequilibrium mapping

Refractive error is a highly heritable quantitative trait responsible for considerable morbidity. Following an initial genome-wide linkage study using microsatellite markers, we confirmed evidence for linkage to chromosome 3q26 and then conducted fine-scale association mapping using high-resolution linkage disequilibrium unit (LDU) maps. We used a preliminary discovery marker set across the 30-Mb region with an average SNP density of 1 SNP/15 kb (Map 1). Map 1 was divided into 51 LDU windows and additional SNPs were genotyped for six regions (Map 2) that showed preliminary evidence of multi-marker association using composite likelihood. A total of 575 cases and controls selected from the tails of the trait distribution were genotyped for the discovery sample. Malecot model estimates indicate three loci with putative common functional variants centred on MFN1 (180,566 kb; 95% confidence interval 180,505–180, 655 kb), approximately 156 kb upstream from alternate-splicing SOX2OT (182,595 kb; 95% CI 182,533–182,688 kb) and PSARL (184,386 kb; 95% CI 184,356–184,411 kb), with the loci showing modest to strong evidence of association for the Map 2 discovery samples (p<10⁻⁷, p<10⁻¹⁰, and p=0.01, respectively). Using an unselected independent sample of 1,430 individuals, results replicated for the MFN1 (p=0.006), SOX2OT (p=0.0002), and PSARL (p=0.0005) gene regions. MFN1 and PSARL both interact with OPA1 to regulate mitochondrial fusion and the inhibition of mitochondrial-led apoptosis, respectively. That two mitochondrial regulatory processes in the retina are implicated in the aetiology of myopia is surprising and is likely to provide novel insight into the molecular genetic basis of common myopia.     

Pex14 is the sole component of the peroxisomal translocon that is required for pexophagy

Pex14 was initially identified as a peroxisomal membrane protein that is involved in docking of the soluble receptor proteins Pex5 and Pex7, which are required for import of PTS1- or PTS2-containing peroxisomal matrix proteins. However, Hansenula polymorpha Pex14 is also required for selective degradation of peroxisomes (pexophagy). Previously we showed that Pex1, Pex4, Pex6 and Pex8 are not required for this process. Here we show that also in the absence of various other peroxins, namely Pex2, Pex10, Pex12, Pex13 and Pex17, pexophagy can normally occur. These peroxins are, like Pex14, components of the peroxisomal translocon. Our data confirm that Pex14 is the sole peroxin that has a unique dual function in two apparent opposite processes, namely peroxisome formation and selective degradation.     

Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as potent HCV polymerase inhibitors

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.     

Environmental heterogeneity generates fluctuating selection on a secondary sexual trait

In any population in which resources are limiting, the allocation of resources toward increased reproductive success may generate costs to survival [1-8]. The relationship between a sexually selected trait and fitness will therefore represent a balance between its relative associations with fecundity versus viability [3, 6, 7]. Because the risk of mortality in a population is likely to be heavily determined by ecological conditions, survival costs may vary as a function of the prevailing environment [7]. As a result, for populations experiencing heterogeneous ecological conditions, there may not be a single optimal level of allocation toward reproduction versus survival [9]. Here, we show that early viability and fecundity selection act in opposing directions on a secondary sexual trait and that their relative magnitude depends upon ecological conditions, generating fluctuating selection. In a wild population of Soay sheep (Ovis aries), phenotypic and genetic associations between male horn growth and lifetime reproductive success were positive under good environmental conditions (because of increased breeding success) and negative under poor environmental conditions (because of reduced survival). In an unpredictable environment, high allocation to early horn growth is a gamble that will only pay off if ensuing conditions are favorable. Such fluctuating selection may play an important role in preventing the erosion of genetic variance in secondary sexual traits.     

Testing for genetic trade-offs between early- and late-life reproduction in a wild red deer population

The antagonistic pleiotropy (AP) theory of ageing predicts genetically based trade-offs between investment in reproduction in early life and survival and performance in later life. Laboratory-based research has shown that such genetic trade-offs exist, but little is currently known about their prevalence in natural populations. We used random regression 'animal model' techniques to test the genetic basis of trade-offs between early-life fecundity (ELF) and maternal performance in late life in a wild population of red deer (Cervus elaphus) on the Isle of Rum, Scotland. Significant genetic variation for both ageing rates in a key maternal performance measure (offspring birth weight) and ELF was present in this population. We found some evidence for a negative genetic covariance between the rate of ageing in offspring birth weight and ELF, and also for a negative environmental covariance. Our results suggest rare support for the AP theory of ageing from a wild population.     

In vivo evidence of role of bone morphogenetic protein-4 in the mouse ovary

The transition of a primordial follicle to a primary follicle is an early step in folliculogenesis. All female mammals are born with a fixed stock of primordial follicles, and exhaustion of that stock leads to menopause or infertility. Recently, several in vitro studies have indicated that BMP-4, BMP-7, and several other growth factors affect the transition of primordial to primary follicles. The aim of our present study was to investigate role of BMP-4 in this process using passive immunization to investigate the role of BMP-4 in a prepubertal mouse model. After seven days of treatment, the weight of antiBMP-4 treated ovaries was significantly lower than the ovaries from mice treated with nonimmune Ig. The number of primary follicles was lower, and the numbers of primordial follicles were higher in antiBMP-4 treated ovaries compared to control ovaries. Treatment with equine chorionic gonadotrophin (eCG) showed no influence on the effects of antiBMP-4 in the mouse ovary. Thus, the results of our study indicate that in vivo BMP-4 acts as transition factor in transition of primordial to primary follicle.     

Protein-protein docking by simulating the process of association subject to biochemical constraints

We present a computational procedure for modeling protein-protein association and predicting the structures of protein-protein complexes. The initial sampling stage is based on an efficient Brownian dynamics algorithm that mimics the physical process of diffusional association. Relevant biochemical data can be directly incorporated as distance constraints at this stage. The docked configurations are then grouped with a hierarchical clustering algorithm into ensembles that represent potential protein-protein encounter complexes. Flexible refinement of selected representative structures is done by molecular dynamics simulation. The protein-protein docking procedure was thoroughly tested on 10 structurally and functionally diverse protein-protein complexes. Starting from X-ray crystal structures of the unbound proteins, in 9 out of 10 cases it yields structures of protein-protein complexes close to those determined experimentally with the percentage of correct contacts >30% and interface backbone RMSD <4 A. Detailed examination of all the docking cases gives insights into important determinants of the performance of the computational approach in modeling protein-protein association and predicting of protein-protein complex structures.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

Myricitrin as a substrate and inhibitor of myeloperoxidase: implications for the pharmacological effects of flavonoids

Flavonoids are increasingly being ingested by the general population as chemotherapeutic and anti-inflammatory agents. They are potentially toxic because of their conversion to free radicals and reactive quinones by peroxidases. Little detailed information is available on how flavonoids interact with myeloperoxidase, which is the predominant peroxidase present at sites of inflammation. This enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, as well as to produce an array of reactive free radicals from organic substrates. We investigated how the flavonoid myricitrin is oxidized by myeloperoxidase and how it affects the activities of this enzyme. Myricitrin was readily oxidized by myeloperoxidase in the presence of hydrogen peroxide. Its main oxidation product was a dimer that underwent further oxidation. In the presence of glutathione, myricitrin was oxidized to a hydroquinone that was conjugated to glutathione. When myeloperoxidase oxidized myricitrin and related flavonoids it became irreversibly inactivated. The number of hydroxyl groups in the B ring of the flavonoids and the presence of a free hydroxyl m-phenol group in the A ring were important for the inhibitory effects. Less enzyme inactivation occurred in the presence of chloride. Neutrophils also oxidized myricitrin to dimers in a reaction that was partially dependent on myeloperoxidase. Myricitrin did not affect the production of hypochlorous acid by neutrophils. We conclude that myricitrin will be oxidized by neutrophils at sites of inflammation to produce reactive free radicals and quinones. It is unlikely to affect hypochlorous acid production by neutrophils.