Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Rapid Parallel Adaptation to Anthropogenic Heavy Metal Pollution

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.     

Availability and Quality of Coronary Heart Disease Family History in Primary Care Medical Records: Implications for Cardiovascular Risk Assessment

Background

The potential to use data on family history of premature disease to assess disease risk is increasingly recognised, particularly in scoring risk for coronary heart disease (CHD). However the quality of family health information in primary care records is unclear.

Aim

To assess the availability and quality of family history of CHD documented in electronic primary care records

Design

Cross-sectional study

Setting

537 UK family practices contributing to The Health Improvement Network database.

Method

Data were obtained from patients aged 20 years or more, registered with their current practice between 1^st January 1998 and 31^st December 2008, for at least one year. The availability and quality of recorded CHD family history was assessed using multilevel logistic and ordinal logistic regression respectively.

Results

In a cross-section of 1,504,535 patients, 19% had a positive or negative family history of CHD recorded. Multilevel logistic regression showed patients aged 50–59 had higher odds of having their family history recorded compared to those aged 20–29 (OR:1.23 (1.21 to 1.25)), however most deprived patients had lower odds compared to those least deprived (OR: 0.86 (0.85 to 0.88)). Of the 140,058 patients with a positive family history recorded (9% of total cohort), age of onset was available in 45%; with data specifying both age of onset and relative affected available in only 11% of records. Multilevel ordinal logistic regression confirmed no statistical association between the quality of family history recording and age, gender, deprivation and year of registration.

Conclusion

Family history of CHD is documented in a small proportion of primary care records; and where positive family history is documented the details are insufficient to assess familial risk or populate cardiovascular risk assessment tools. Data capture needs to be improved particularly for more disadvantaged patients who may be most likely to benefit from CHD risk assessment.     

Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis

Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss‐of‐function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence‐based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.     

Prenatal exposure to chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing germ cell cyst breakdown in the F1 offspring

Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries such as chrome plating, welding, wood processing and tanneries. As one of the world?s leading producers of chromium compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. CrVI is rapidly converted to CrIII intracellularly, and can induce apoptosis through different mechanisms. Our previous studies demonstrated postnatal exposure to CrVI results in a delay or arrest in follicle development and puberty. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD 20, and total Cr was estimated in the placentae; ovaries were removed from the F1 offspring on postnatal day (PND)-1 and various analyses were performed. Our results show that gestational exposure to CrVI resulted in (i) increased Cr concentration in the placenta, (ii) increased germ cell apoptosis by up-regulating p53/p27–Bax–caspase-3 proteins and by increasing p53–SOD-2 co-localization; (iii) accelerated germ cell cyst (GCC) breakdown; (iv) advanced primordial follicle assembly and primary follicle transition and (v) down regulation of p-AKT, p-ERK and XIAP. As a result of the above events, CrVI induced early reproductive senescence and decrease in litter size in F1 female progeny.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Chromosomes of antelope squirrels (genus Ammospermophilus): A systematic banding analysis of four species with unusual constitutive heterochromatin

The G- and C-banding patterns of mitotic chromosomes from four species of antelope squirrels (Ammospermophilus harrisi, interpres, leucurus and nelsoni) are discussed with special attention payed to the unusual quantities and position of constitutive heterochromatin. Heterochromatin appears to be responsible for the observation that cells from antelope squirrels contain over 70% more DNA than cells from another ground squirrel. A substantial fraction of this excess DNA consists of sequences that band as satellites in neutral CsCl or Cs₂SO₄-Ag⁺ density gradients. Interspecies similarities in the distribution of heterochromatin suggest that it has a function of some importance to these species and has therefore been conserved.     

Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.