Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Transient ipsilateral retinal ganglion cell projections to the brain: Extent, targeting, and disappearance

Author ManuscriptDuring development of the mammalian eye, the first retinal ganglion cells (RGCs) that extend to the brain are located in the dorsocentral (DC) retina. These RGCs extend to either ipsilateral or contralateral targets, but the ipsilateral projections do not survive into postnatal periods. The function and means of disappearance of the transient ipsilateral projection are not known. We have followed the course of this transient early ipsilateral cohort of RGCs, paying attention to how far they extend, whether they enter targets and if so, which ones, and the time course of their disappearance. The DC ipsilateral RGC axons were traced using DiI labeling at E13.5 and E15.5 to compare the proportion of ipsi- versus contralateral projections during the first period of growth. In utero electroporation of E12.5 retina with GFP constructs was used to label axons that could be visualized at succeeding time points into postnatal ages. Our results show that the earliest ipsilateral axons grow along the cellular border of the brain, and are segregated from the laterally positioned contralateral axons from the same retinal origin. In agreement with previous reports, although many early RGCs extend ipsilaterally, after E16 their number rapidly declines. Nonetheless, some ipsilateral axons from the DC retina enter the superior colliculus and arborize minimally, but very few enter the dorsal lateral geniculate nucleus and those that do extend only short branches. While the mechanism of selective axonal disappearance remains elusive, these data give further insight into establishment of the visual pathways.Contract grant sponsor: NIH (C.A.M.); contract grant number: R01 EY012736 and P30 EY019007.Contract grant sponsors: Portuguese Foundation for Science and Technology fellowship SFRH/BD/74926/2010; Luso-American Development Foundation; MD-PhD Program, University of Minho (C.A.S.)

Depth of reading vocabulary in hearing and hearing-impaired children

The main point of our study was to examine the vocabulary knowledge of pupils in grades 3–6, and in particular the relative reading vocabulary disadvantage of hearing-impaired pupils. The achievements of 394 pupils with normal hearing and 106 pupils with a hearing impairment were examined on two vocabulary assessment tasks: a lexical decision task and a use decision task. The target words in both tasks represent the vocabulary children should have at the end of primary school. The results showed that most hearing pupils reached this norm, whereas most hearing-impaired pupils did not. In addition, results showed that hearing-impaired pupils not only knew fewer words, but that they also knew them less well. This lack of deeper knowledge remained even when matching hearing and hearing-impaired children on minimal word knowledge. Additionally, comparison of the two tasks demonstrated the efficacy of the lexical decision task as a measure of lexical semantic knowledge

Preparation and Characterization of a Lovastatin-Loaded Protein-Free Nanostructured Lipid Carrier Resembling High-Density Lipoprotein and Evaluation of its Targeting to Foam Cells

This study was designed to investigate whether a non-protein nanostructured lipid carrier (NLC) resembling high-density lipoprotein (HDL) could deliver a hydrophobic anti-atherogenic drug, lovastatin, to foam cells. Lovastatin-loaded NLC (LT-NLC) was prepared by a nanoprecipitation/solvent diffusion method. The LT-NLC-apoprotein (LT-NLC-apo) was prepared by incubating LT-NLC with native HDL. The physicochemical parameters of LT-NLC were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, and crystallization behavior. Targeting behavior and mechanism were demonstrated by the incubation of LT-NLC-apo with a RAW 264.7 macrophage-derived foam cell model in the presence or absence of very-low-density lipoprotein (VLDL) and lipase. The results showed that LT-NLC was solid spherical or oval in shape with an average diameter of 13.8 ± 2.2 nm, zeta potential of −29.3 ± 0.2 mV and entrapment efficiency of 96.2 ± 1.3%. Phagocytosis studies showed that uptake of LT-NLC-apo by macrophages was significantly lower than LT-NLC (p < 0.01), suggesting that LT-NLC-apo could possibly escape recognition from macrophages in vivo. The uptake was increased twofold when LT-NLC-apo was incubated with transfected foam cells containing VLDL and lipase. These results indicated that non-protein NLC resembling HDL could be a useful tool to deliver lipophilic anti-atherogenic drugs to foam cells, and that uptake could be enhanced by the VLDL receptor pathway

Manganese superoxide dismutase Ala-9Val polymorphism and risk of breast cancer in a population-based case–control study of African Americans and whites

INTRODUCTION: A polymorphism in the manganese superoxide dismutase (MnSOD) gene, Ala-9Val, has been examined in association with breast cancer risk in several epidemiologic studies. Results suggest that the Ala allele increases the risk of breast cancer and modifies the effects of environmental exposures that produce oxidative damage to DNA. METHODS: We examined the role of the MnSOD Ala-9Val polymorphism in a population-based case–control study of invasive and in situ breast cancer in North Carolina. Genotypes were evaluated for 2025 cases (760 African Americans and 1265 whites) and for 1812 controls (677 African Americans and 1135 whites). RESULTS: The odds ratio for MnSOD Ala/Ala versus any MnSOD Val genotypes was not elevated in African Americans (odds ratio = 0.9, 95% confidence interval = 0.7–1.2) or in whites (odds ratio = 1.0, 95% confidence interval = 0.8–1.2). Greater than additive joint effects were observed for the Ala/Ala genotype and smoking, radiation to the chest, and occupational exposure to ionizing radiation. Antagonism was observed between the Ala/Ala genotype and the use of nonsteroidal anti-inflammatory drugs. CONCLUSIONS: The MnSOD genotype may contribute to an increased risk of breast cancer in the presence of specific environmental exposures. These results provide further evidence for the importance of reactive oxygen species and of oxidative DNA damage in the etiology of breast cancer

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Environmental Factors Affecting Large-Bodied Coral Reef Fish Assemblages in the Mariana Archipelago

Large-bodied reef fishes represent an economically and ecologically important segment of the coral reef fish assemblage. Many of these individuals supply the bulk of the reproductive output for their population and have a disproportionate effect on their environment (e.g. as apex predators or bioeroding herbivores). Large-bodied reef fishes also tend to be at greatest risk of overfishing, and their loss can result in a myriad of either cascading (direct) or indirect trophic and other effects. While many studies have investigated habitat characteristics affecting populations of small-bodied reef fishes, few have explored the relationship between large-bodied species and their environment. Here, we describe the distribution of the large-bodied reef fishes in the Mariana Archipelago with an emphasis on the environmental factors associated with their distribution. Of the factors considered in this study, a negative association with human population density showed the highest relative influence on the distribution of large-bodied reef fishes; however, depth, water temperature, and distance to deep water also were important. These findings provide new information on the ecology of large-bodied reef fishes can inform discussions concerning essential fish habitat and ecosystem-based management for these species and highlight important knowledge gaps worthy of additional research

A population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients

INTRODUCTION: The Oncotype DX assay was recently reported to predict risk for distant recurrence among a clinical trial population of tamoxifen-treated patients with lymph node-negative, estrogen receptor (ER)-positive breast cancer. To confirm and extend these findings, we evaluated the performance of this 21-gene assay among node-negative patients from a community hospital setting. METHODS: A case-control study was conducted among 4,964 Kaiser Permanente patients diagnosed with node-negative invasive breast cancer from 1985 to 1994 and not treated with adjuvant chemotherapy. Cases (n = 220) were patients who died from breast cancer. Controls (n = 570) were breast cancer patients who were individually matched to cases with respect to age, race, adjuvant tamoxifen, medical facility and diagnosis year, and were alive at the date of death of their matched case. Using an RT-PCR assay, archived tumor tissues were analyzed for expression levels of 16 cancer-related and five reference genes, and a summary risk score (the Recurrence Score) was calculated for each patient. Conditional logistic regression methods were used to estimate the association between risk of breast cancer death and Recurrence Score. RESULTS: After adjusting for tumor size and grade, the Recurrence Score was associated with risk of breast cancer death in ER-positive, tamoxifen-treated and -untreated patients (P = 0.003 and P = 0.03, respectively). At 10 years, the risks for breast cancer death in ER-positive, tamoxifen-treated patients were 2.8% (95% confidence interval [CI] 1.7–3.9%), 10.7% (95% CI 6.3–14.9%), and 15.5% (95% CI 7.6–22.8%) for those in the low, intermediate and high risk Recurrence Score groups, respectively. They were 6.2% (95% CI 4.5–7.9%), 17.8% (95% CI 11.8–23.3%), and 19.9% (95% CI 14.2–25.2%) for ER-positive patients not treated with tamoxifen. In both the tamoxifen-treated and -untreated groups, approximately 50% of patients had low risk Recurrence Score values. CONCLUSION: In this large, population-based study of lymph node-negative patients not treated with chemotherapy, the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive, tamoxifen-treated and -untreated patients

Cigarette smoking, genetic polymorphisms and colorectal cancer risk: the Fukuoka Colorectal Cancer Study

Background: It is uncertain whether smoking is related to colorectal cancer risk. Cytochrome P-450 CYP1A1, glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are important enzymes in the metabolism of tobacco carcinogens, and functional genetic polymorphisms are known for these enzymes. We investigated the relation of cigarette smoking and related genetic polymorphisms to colorectal cancer risk, with special reference to the interaction between smoking and genetic polymorphism. Methods: We used data from the Fukuoka Colorectal Cancer Study, a population-based case-control study, including 685 cases and 778 controls who gave informed consent to genetic analysis. Interview was conducted to assess lifestyle factors, and DNA was extracted from buffy coat. Results: In comparison with lifelong nonsmokers, the odds ratios (OR) of colorectal cancer for &lt;400, 400-799 and ≥800 cigarette-years were 0.65 (95 % confidence interval [CI], 0.45-0.89), 1.16 (0.83-1.62) and 1.14 (0.73-1.77), respectively. A decreased risk associated with light smoking was observed only for colon cancer, and rectal cancer showed an increased risk among those with ≥400 cigarette-years (OR 1.60, 95 % CI 1.04-2.45). None of the polymorphisms under study was singly associated with colorectal cancer risk. Of the gene-gene interactions studied, the composite genotype of CYP1A1*2A or CYP1A1*2C and GSTT1 polymorphisms was associated with a decreased risk of colorecta